http://2010.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Georgio2010.igem.org - User contributions [en]2024-03-28T14:37:28ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:ESBS-Strasbourg/proteolux/sitemapTeam:ESBS-Strasbourg/proteolux/sitemap2010-11-30T13:13:20Z<p>Georgio: </p>
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<div>{{ESBS-Strasbourg/Temp2}} {|<br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Proteolux.swfFile:ESBS-Strasbourg-Proteolux.swf2010-11-30T13:11:44Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/sitemapTeam:ESBS-Strasbourg/proteolux/sitemap2010-11-30T12:43:08Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<div>{{ESBS-Strasbourg/Temp2}} {|<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/contactTeam:ESBS-Strasbourg/proteolux/contact2010-11-30T12:40:08Z<p>Georgio: </p>
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/overview/proteoluxbasic2010-11-30T12:31:38Z<p>Georgio: </p>
<hr />
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<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<br />
<br />
</li><br />
<br />
<br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br /><br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
<br />
<br />
</ul><br />
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<br><br />
<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
</ul><br />
<br />
</div><br />
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<div class="desc"><br />
<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
<br />
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<div class="heading"><br />
<a name="Animation"></a><br />
Proteolux Plus ®<br />
</div><br />
<br><br />
<br><br />
<center><br />
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<td width="750" bgcolor="#414141" valign="top"><br />
<div class="heading"><br />
<a name="Animation"></a><br />
Proteolux Plus ®<br />
</div><br />
<br><br />
<br><br />
<center><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxproTeam:ESBS-Strasbourg/proteolux/overview/proteoluxpro2010-11-30T12:29:47Z<p>Georgio: </p>
<hr />
<div>{{ESBS-Strasbourg/Temp2}} {|<br />
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<div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"><br />
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/contact" style="text-decoration: none"><br />
<font color="#FFFFFF">Contact</font></a>&nbsp;&nbsp;&nbsp;<br />
<a style="text-decoration: none"><br />
<font color="#FFFFFF">|</font></a>&nbsp;&nbsp;&nbsp;<br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/sitemap" style="text-decoration: none"><br />
<font color="#FFFFFF">Sitemap</font></a><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
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<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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<a name="Animation"></a><br />
Proteolux Plus ®<br />
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<center><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/K5jTzhzREic?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/K5jTzhzREic?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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<a name="Animation"></a><br />
Proteolux Plus ®<br />
</div><br />
<br><br />
<br><br />
<center><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/pvdjXNAQMIM?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/pvdjXNAQMIM?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplusTeam:ESBS-Strasbourg/proteolux/overview/proteoluxplus2010-11-30T12:28:28Z<p>Georgio: </p>
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<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br /><br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
<br />
<br />
</ul><br />
<br />
</div><br />
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<br />
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<br><br />
<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
</ul><br />
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<div class="desc"><br />
<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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Proteolux Plus ®<br />
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Proteolux Plus ®<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/contactTeam:ESBS-Strasbourg/proteolux/contact2010-11-30T10:35:19Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
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<div class="heading">ProteOlux ®</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
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Team<br />
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<font size="1">Figure 3: Light controlled intrabody formation and specific target protein degradation.<br />
A: Structure of an IgG antibody. Shown are the two long heavy (H) chains and the small light (L) chains. The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The chains are intra- and interconnected by disulfide bridges (yellow lines). B1.1 and B1.2: Fusion constructs for the light and heavy chains. B1.1: The blue light photoreceptor Zeitlupe (ZTL), the Phytochrome interacting factor (PIF) and the C-terminal degradation tag DAS are fused to the C-terminus of the light chain. B1.2: The gigantea (GI) sequence is fused in the hinge region of the antibody. The two conserved cysteine residues for the disulfide bridge are maintained, the two constant domains (CH2 and CH3) of the heavy chain are deleted. C: Blue light dependent photo conversion of ZTL. The absorption of blue light (475 nm) by the PAS-like LOV domain of ZTL leads to a conformational change allowing GI binding (D). D: Blue light induced formation of the active intrabody. The binding of GI to ZTL under blue light brings the light and heavy chain together and restores the natural disulfide bridge between the two. The two variable domains can now form an active antigen binding site that binds to the protein of interest. E: Red light induced protein degradation. PIF binds to PhyB under red light conditions bringing the DAS tag in proximity to the ClpXP protease leading to degradation of the protein of interest bound to the intrabody.</font></div><br />
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<p><b>Light-controlled regulation of the intrabody activity.</b></p><br />
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The idea is to have an inactive intrabody which can be assembled on demand into its active form. Therefore, the structure of a general IgG antibody has to be regarded in greater detail (figure 1) [Garrett RH and Grisham CM, Biochemistry 2nd edition].<br />
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<p><span><font size="1">Figure 1: Overview over the structure of an IgG antibody. The active IgG antibody is composed of two heavy (H) chains (outer long chains) and the small light (L) chains (inner short chains). The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The Fab region which is composed of the variable domain and the CH1 domain is connected via a flexible hinge region to the Fc part of the antibody (CH2 and CH3 region). The chains are intra- and interconnected by disulfide bridges (yellow lines). There are two disulfide bridges that interconnect the two heavy chains and each variable chain is connected to one heavy chain via one disulfide bridge. The active antibody is only formed when the light chain is connected to the heavy chain in order to form the antigen binding site, which is situated between the variable domains. </font></span></p><br />
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The formation of the antigen binding site is dependent on the correct binding of the VL to the VH domain, which is enhanced by a strong affinity between the constant regions and the disulfide bridge between the light and the heavy chain. In the Proteolux Pro system, this permanent disulfide bridge is replaced by a light inducible interaction between two proteins. In order to avoid interference with the PhyB-PIF interaction, other interaction partners were searched which are active under light conditions that do not activate phytrochrome B (figure 2). <br />
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<p><span><font size="1">Figure 2: Absorption spectrum of phytrochome B containing the PCB chromophore. The absorption maxima of the two red light forms are visible as two distinct peaks at 658 nm for the red light form and at 720 nm for the far-red light form. The absorbance is lowest in the blue/green light part of the spectrum between 450nm and 550nm. Therefore, it is save to work under blue/green light conditions to avoid the activation of phytrochrome B. </font></span></p><br />
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The absorbance spectrum of phytrochrome B reveals that it does not absorb under light conditions between 450 nm and 550 nm which correlates to blue/green light. Therefore, the blue light (475 nm) regulated interaction between the Arabidopsis thaliana photoreceptor Zeitlupe (ZTL, Gene ID: 835842) and Gigantea (GI, Gene ID: 838883) is used to replace the disulfide bridge function. Zeitlupe contains a light sensing PAS-like LOV domain at its N-terminal end and a C-terminal Kelch repeat domain, which is involved in protein-protein interaction [Lariguet P and Dunand C, 2005; http://www.uniprot.org/uniprot/Q94BT6]. The GI protein has a more than 4 fold affinity to ZTL under blue light conditions [Kim WY, 2007].<br><br />
In order to prevent the strong affinity between the constant domains of the light and the heavy chain, only the low affinity variable domains are used in the construction. <br><br />
The ZTL sequence is fused together with the normal PIF-DAS-tag to the C-terminus of the VL domain and the GI sequence followed by a GST sequence to the C-terminus of the VH domain. The GST dimerizes and connects therefore two VH domains. This allows the formation of bivalent intrabodies with higher protein binding capacity.<br><br />
The expression of such a modified intrabody allows the formation of the active intrabody only under blue light conditions. The protein expressing phenotype can be analyzed under far-red light conditions. When the protein should be degraded, the intrabody formation is triggered under blue light (475nm) resulting in the formation of an active antibody binding site and fixation of the target protein by the intrabody. To ensure, that the protein is not active anymore the intrabody-protein complex can be tethered to the protease using red light conditions. This procedure is summarized in figure 3. <br><br><br />
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<p><b>Proteolux Pro offers the first light-controlled intrabody activity which can be used to analyze any protein in its native conformation without interference with the cell metabolism. </b></p></center><br />
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<center><br />
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<div style="position: relative; width: 650px; height: 150px; id="layer1" align="justify"><br />
<font size="1">Figure 3: Light controlled intrabody formation and specific target protein degradation.<br />
A: Structure of an IgG antibody. Shown are the two long heavy (H) chains and the small light (L) chains. The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The chains are intra- and interconnected by disulfide bridges (yellow lines). B1.1 and B1.2: Fusion constructs for the light and heavy chains. B1.1: The blue light photoreceptor Zeitlupe (ZTL), the Phytochrome interacting factor (PIF) and the C-terminal degradation tag DAS are fused to the C-terminus of the light chain. B1.2: The gigantea (GI) sequence is fused in the hinge region of the antibody. The two conserved cysteine residues for the disulfide bridge are maintained, the two constant domains (CH2 and CH3) of the heavy chain are deleted. C: Blue light dependent photo conversion of ZTL. The absorption of blue light (475 nm) by the PAS-like LOV domain of ZTL leads to a conformational change allowing GI binding (D). D: Blue light induced formation of the active intrabody. The binding of GI to ZTL under blue light brings the light and heavy chain together and restores the natural disulfide bridge between the two. The two variable domains can now form an active antigen binding site that binds to the protein of interest. E: Red light induced protein degradation. PIF binds to PhyB under red light conditions bringing the DAS tag in proximity to the ClpXP protease leading to degradation of the protein of interest bound to the intrabody.</font></div><br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Team.pngFile:ESBS-Strasbourg-Team.png2010-11-30T10:32:16Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-30T10:31:50Z<p>Georgio: </p>
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#degradation">Degradation System</a></li><br />
<li><a href="#light">Light detection system</a></li><br />
<li><a href="#system">Final construction</a></li><br />
<li><a href="#chassis">Introduction to various chassis</a></li><br />
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<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
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The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
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PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
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Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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Degradation system<br />
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<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
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<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
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To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
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<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
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<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
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Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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Light detection system<br />
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As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
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<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
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All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
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<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
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The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
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We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
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<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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Final construction: Light controllable protease <br />
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PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
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The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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<a name="chassis"></a><br />
Introduction to various chassis <br />
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The ProteoLux® system can be applied to various chassis. To optimize the expression we developed different versions of ProteoLux® based on an improved codon usage especially adapted to the target host system : <br />
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<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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<b>ProteoLux® bacteria</b> has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
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<b>ProteoLux® bacteria</b> has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
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<b>ProteoLux® eucaria</b> has been designed for the use in eukaryotic microorganism like Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organism in molecular and cellular biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
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<b>ProteoLux® eucaria</b> has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
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<b>ProteoLux® mammalia</b> aims at the use in the medical research field, its codon usage has been adapted to the human species. <br />
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The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
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For in vivo implementations you should consider the use ProteoLux® Pro, which presents a special variant of ProteoLux® mammalia. <br />
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The introduction of ProteoLux® can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria. <br />
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Mammalian species also have a ClpXP but it is expressed in mitochondria. Thus, the natural protease will not interfere with the ProteoLux® protease. Besides, it has be shown that the human ClpXP does not recognize SsrA tag and lambda Otag (Functional Proteolytic Complexes of the Human Mitochondrial ATP-dependent Protease, hClpXP (2002) J. Biol. Chem. Vol. 277, No. 23, pp. 21095–21102).<br />
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<br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
<br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
<br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of ProteoLux®, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
<br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign" style="text-decoration: none"> <font color="#FF9900">targeting construct design.</font></a><br />
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<a name="scheme"></a><br />
SCHEME <br />
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To avoid this time consuming step you can resort to the ProteoLux® Pro system which further has lots of potential for in vivo studies. The details of this promising approach are explained in the following chapter. <br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancerTeam:ESBS-Strasbourg/proteolux/application/cancer2010-11-30T10:28:01Z<p>Georgio: </p>
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<ul><br />
<li><a href="#cancer">Cancer</a></li><br />
<li><a href="#pancreatic">Pancreatic Ductal Adenocarcinoma</a></li><br />
<li><a href="#molecular">Molecular Pathogenesis of PDAC</a></li><br />
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<a name="cancer"></a><br />
ProteoLux® Pro system against cancer<br />
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<b>Proteomics</b> technology is an approach of science to understand the expression of the whole set of proteins and its function at the cellular level. Its role in cancer research becomes more significant nowadays. Consulting Medline reveals that, since 1997, when the word proteomics first appeared as an entry, approx. 0.12% of all articles on cancer also contain the word proteomics. Comparing this frequency with the occurrence of the search string ‘genomics’ in 0.17% of the cancer literature, it becomes obvious that proteomics has attracted major scientific attention in the field of cancer research. Vice versa, a search for ‘cancer AND proteomics’ shows that 16% of the proteomics literature deals with cancer, testifying that the attraction is mutual. <br />
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Indeed, proteomics technology enables the researchers to look into the proteins level of the cancer cells, proteins being the physiological and pathological indispensible players. Proteins represent the majority of drug targets. However, currently only around 500 out of the estimated >3000 proteins that are predicted to be druggable are exploited.<br />
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<b>Proteomics has the great potential to be one of the most powerful tools for cancer research.</b> The <b>ProteoLux® Pro system offers a new proteomic tool for cancer research.</b> It can help in therapy designing by characterizing the potency of drug targets. The multiple targets of the system and its advantages will be exemplified in the case of the pancreatic ductal adenocarcinoma (PDAC), a major unsolved health problem.<br />
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<a name="pancreatic"></a><br />
Pancreatic ductal adenocarcinoma (PDAC)<br />
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Pancreas is a gland organ found in vertebrates. The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. It presents endocrine and exocrine functions. In fact this gland permits to produce hormones like insulin and to secrete digestive enzymes contained in a pancreatic juice which are inserted into the small intestine to help for assimilation of nutrients. The exocrine pancreas consists of acinar and duct cells. The Figure 12 describes in detail the pancreas anatomy. <br />
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<img src="https://static.igem.org/mediawiki/2010/5/57/ESBS-Strasbourg-pancreas1.png" width="400px" height="313px"></a><br />
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<center><br />
<div style="position: relative; width: 400px; height: 220px; id="layer1" align="justify"><br />
<font size="1">Figure 1 | Anatomy of the pancreas.<br><br />
The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. The exocrine pancreas consists of acinar and duct cells. The acinar cells produce digestive enzymes and constitute the bulk of the pancreatic tissue. They are organized into grape-like clusters that are at the smallest termini of the branching duct system. The ducts, which add mucous and bicarbonate to the enzyme mixture, form a network of increasing size, culminating in main and accessory pancreatic ducts that empty into the duodenum. The endocrine pancreas, consisting of four specialized cell types that are organized into compact islets embedded within acinar tissue, secretes hormones into the bloodstream. The α- and β-cells regulate the usage of glucose through the production of glucagon and insulin, respectively. Pancreatic polypeptide and somatostatin that are produced in the PP and δ-cells modulate the secretory properties of the other pancreatic cell types. a | Gross anatomy of the pancreas. b | The exocrine pancreas. c | A single acinus. d | A pancreatic islet embedded in exocrine tissue.<br><br />
Bardeesy N and DePinho R A. (2002) ”PANCREATIC CANCER BIOLOGY AND GENETICS” Nature Reviews Cancer VOL.2 pp897-909<br />
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Pancreatic Cancer is a cancer of the digestive system. Different kinds of pancreatic cancer exist : Adenocarcinomas, which represents 95% of cases, adenosquamous carcinomas, singet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas and undifferentiated carcinomas with osteoclast-like giant cells which represent the remaining 5%. <br />
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<font size="1">Figure 2 | Gross photograph of an infiltrating adenocarcinoma<br><br />
Note the dramatic narrowing of the pancreatic duct associated with the poorly defined white neoplasm.<br><br />
Maitra A, 1,2 and Ralph H. Hruban R H. (2008) “Pancreatic cancer” Annu. Rev. Pathol. Mech. Dis. 3:157–88<br />
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<b>Pancreatic ductal adenocarcinoma</b> is a malignant tumor of epithelia originating in glandular tissue. It grossly produces a firm, highly sclerotic mass (Figure 2). <b>Aggressive and devastating disease</b>, it is one of the most common causes of cancer related death. It is characterized by invasiveness, rapid progression and <b>profound resistance to treatment.</b><br />
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<img src="https://static.igem.org/mediawiki/2010/9/91/ESBS-Strasbourg-Pancreas3.png" width="250px" height="239px"></a><br />
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<div style="position: relative; width: 250px; height: 50px; id="layer1" align="justify"><br />
<font size="1">Figure 3 | Mortality of the pancreas cancer per year<br><br />
<a href="http://commons.wikimedia.org/wiki/File:Pancreas_cancer_world_map_-_Death_-_WHO2004.svg">link</a><br />
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Pancreatic cancer is predominantly a disease of the elderly. Besides, men are more concerned than women. Age and gender are so risk factors. Other established risk factors include diets high in meats and fat, low serum folate levels, obesity, long-standing diabetes mellitus, and chronic pancreatitis. Pancreatic cancer may be genetic. This is the case for 5 to 10% of patient but the gene responsible of this disease is not yet identified. Furthermore for the moment there are no real guidelines for preventing from pancreatic cancer.<br />
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Developed countries are the most affected by this disease as shown in Figure 3. <b>This cancer causes the death of thousands people in the world each year</b> of whom 7000 French and 36 800 American.<br />
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This cancer is <b>very hard to detect.</b> Indeed, there is often no symptom attached to early pancreatic cancer. Furthermore the later symptoms are non specific and varied. That is why this disease is also called “silent killer”. Pain in the abdomen, loss of appetite and so loss of weight, painless jaundice, diabetes mellitus or elevated blood sugar level, trousseau sign or clinical depression are common symptoms of pancreatic cancer. <br />
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The <b>prognosis</b> of the pancreatic cancer is poor. The <b>5- year survival rate</b> of all patients is <b>below 5%</b>, and the median survival time after diagnosis is 6 months. <b>Surgery offered the only possibility of cure.</b> But surgery may only be used if the tumor is not too advanced and if the cancer does not present metastasis. This surgery is not easy because of the high amount of veins surrounding the pancreas and this operation is possible only in 20% of cases. Furthermore a relapse is common (70 to 80% of people). Thus, only 20% of the patients who have undergone surgery survive longer than 5 years. For those who cannot undergo this surgery their median survey is approximately of 6 months. Radiotherapy may be used in case of evolved cancer and is established after surgery. Then chemotherapy may be used when the cancer presents metastasis. Gemcitabine, Fluorouracil, cisplatine and oxaliplatine are used. Nevertheless, <b>PDAC is insensitive to most therapies including chemotherapy, radiotherapy and immunotherapy.</b><br />
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<a name="molecular"></a><br />
Molecular Pathogenesis of PDAC<br />
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<font size="1">Figure 4 |Progression model of pancreatic adenocarcinoma<br><br />
<a href="http://www.nature.com/modpathol/journal/v15/n4/images/3880544f1.jpg + medscape.com">link</a><br />
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The progression from histologically normal epithelium to low-grade pancreatic intraepithelial neoplasia (PanINs are microscopic lesions in the smaller (<5 mm) pancreatic ducts), to high-grade PanIN, to invasive carcinoma (left to right in Figure 4) is associated with the accumulation of specific genetic alterations. This signature molecular profile consists notably in <b>mutations in the oncogene K-RAS and the tumor suppressors CDKN2, TP53, SMAD4/DPC4 and BRCA2.</b> Other recognized precursor lesions of adenocarcinoma (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) likely harbor a distinct compendium of genetic alterations in their path to invasive cancer. <br />
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The Figure 5 presents a schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma.<br />
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<img src="https://static.igem.org/mediawiki/2010/8/8b/ESBS-Strasbourg-Pancreas5.png" width="400px" height="310px"></a><br />
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<div style="position: relative; width: 400px; height: 150px; id="layer1" align="justify"><br />
<font size="1">Figure 5 | Schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma<br><br />
O'Reilly E and Epstein A (2010) “Recent Findings in Pancreatic Cancer: Illuminating Emerging Treatment Strategies” MedscapeCME Oncology <br><br><br />
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EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinases; FGF-R = fibroblast growth factor receptor; GDP = guanosine diphosphate; GTP = guanosine triphosphate; HER2 = human EGFR 2; IGF = insulin growth factor; MEK = mitogen-activated protein kinase; MEKK = mitogen-activated protein kinase kinase; MMP = matrix metalloproteinase; mTOR = mammalian target of rapamycin; NF-kappaB = nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF = vascular endothelial growth factor receptor. <br />
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The <b>majority (90%) of pancreatic adenocarcinomas</b> have a <b>mutated K-Ras oncogene.</b> KRAS encodes a member of the RAS family of guanosine triphosphate (GTP)-binding proteins that mediate a range of cellular functions, including proliferation, cell survival, cytoskeletal remodeling, and motility, among others. Activating mutations impair the intrinsic GTPase activity of the KRAS gene product, resulting in a protein that is constitutively active in intracellular signal transduction. In addition to its role in pancreatic cancer initiation, constitutive RAS signaling appears to be required for pancreatic cancer maintenance as well.<br />
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For many years targeting, Ras has been a holy grail in the treatment of pancreatic cancer. Either single-agent or combination therapies with farnesyl transferase inhibitors (SWOG 9924 study) that target Ras translocation have shown limited activity. <b>Ras</b> still represents an <b>attractive and ubiquitous target.</b><br />
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Activated Kras engages a number of downstream effector pathways, including RAF–mitogen-activated protein kinase (RAFMAPK), phosphoinositide-3-kinase (PI3K), and RalGDS pathways (as shown in Figure 6) producing thus a remarkable array of cellular effects, including induction of proliferation, survival and invasion through the stimulation of several effector pathways. <br />
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<img src="https://static.igem.org/mediawiki/2010/3/39/ESBS-Strasbourg-Pancreas6.png" width="400px" height="292px"></a><br />
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<font size="1">Figure 6 | Some pathways in which KRAS is involved in PDAC<br><br />
Lauth M, Toftgard R and al (2010) “DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS” Nat. Struct. Mol. Biol. 17, 718 - 725<br />
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<p><b>Mirk/Dyrk1B as potential target in pancreas cancer </b></p><br />
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<b>DYRK1B/Mirk</b> is a RAS effector kinase (Figure 6), which is activated by phosphorylation by its upstream activator, the MAP kinase kinase MKK3, suggesting that Mirk, like p38, is activated by certain environmental stress agents. <br />
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Mirk/Dyrk1B is a member of the Minibrain/dyrk family of kinases, more precisely an arginine-directed serine/threonine protein kinase. It is expressed at low levels in most normal tissues but at elevated levels in quiescent pancreatic cancer cells. Mirk is expressed in about 90% of pancreatic cancers and is amplified in a subset. Mirk appears not to be an essential gene for normal cells from embryonic knockout studies in mice and RNA interference studies on cultured cells. Mirk/Dyrk1B mediates the prolonged survival of cancer cells through increasing expression of a cohort of antioxidant genes. These unusual characteristics suggest that Mirk may be a selective target for therapeutic intervention [70].<br />
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Indeed, one major issue in pancreas cancer is the insensitivity to treatment due to the activation of defense systems, notably those producing antioxydants in which Mirk acts. By inhibiting Mirk, the protection of cancer cells will be inhibited, making them more sensitive to actual treatment (chemotherapy, radiotherapy…). <br />
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<p><b>Advantages of the ProteoLux® Pro system in cancer research</b></p><br />
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The <b>ProteoLux® Pro system enable to</b> <font color="#800000"><b>degrade specifically one or more proteins expressed in vivo at a given time and in a given cell type.</b></font><br />
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The <b>ProteoLux® Pro system also enables you to control the concentration of a protein according to the lighting alternation between far red light and red light. The ProteoLux® Pro system also enables you to degrade specifically muted protein (not the normal one) by using specific intrabodies.</b><br />
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<p><b><u>The ProteoLux® Pro system : an amazing tool to study every overexpressed protein, notably oncoproteins.</u></b></p><br />
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Indeed, oncogenes which code for oncoproteins are mutated proto-oncogenes, very important genes involved in the regulation of cell cycle, cell growth and cell proliferation. Mutations in proto-oncogenes can lead:<br />
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<ul><br />
<li>either to a conformational change of the oncoprotein, thus to an increase of its activity or a loss of its regulation</li><br />
<li>or to an increase of its activity, an increase of its stability, a duplication of the oncogene, a retroviral insertion, all this leading to an increase of the concentration of the oncoprotein</li><br />
<li>or to a chromosomic translocation (expression in the wrong cell type, at the wrong time or constitutive expression of the oncogene).</li><br />
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<p><b><u>The ProteoLux® Pro system : a rapid access to the potency of therapeutic targets</u></b></p><br />
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The potency of Mirk as a therapeutic target can easily and relatively fastly be studied with the ProteoLux® system.<br />
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<img src="https://static.igem.org/mediawiki/2010/c/c4/ESBS-Strasbourg-Pancreas7.png" width="400px" height="284px"></a><br />
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<font size="1">Figure 7 | Principle of the assessment of the potency as therapeutic target by the ProteoLux® Pro system<br />
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Besides, by using several intrabodies it is possible to target several proteins at the same time. Therefore, the ProteoLux® Pro system permits an easy assessment of combinatory targeting.<br />
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<p><b><u>The ProteoLux® Pro system : solution to the specific difficulty of clinical management in cancer</u></b></p><br />
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As shown in Figure 4, tumors possess cytogenetically different clones corresponding to different stages of progression of the disease. This heterogeneity contributes to differences in clinical behavior and responses to treatment of tumors of the same diagnostic type. With the ProteoLux® system you can target several muted proteins by intrabodies. Thus, you will only target muted proteins but all the muted proteins. Therefore you will target all the stages of progression of the disease.<br />
<br><br><br />
The discovery and understanding of the altered genes causing cancer (oncogenes, tumor-suppressor genes, and microRNA genes) and of the pathways involved permit the development of cancer therapies. Unfortunately, research goes slowly. The <b>ProteoLux® Pro system gives research a boost.</b><br />
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The ProteoLux® system can interest big pharmaceutical firms like Roche or Novartis which are already focusing on pancreatic cancer. Indeed, Roche has developed a molecule for chemotherapy, Traceva and Novartis has created a new product Afinitor, which permit also to increase life expectancy of people affect by pancreatic endocrine tumor. Our system permits these companies to broaden their scope against pancreatic cancer by studying easily the potency of a new target. Notably, they will be able to develop cancer stage-specific drugs basing on the ProteoLux® system.<br />
<br><br><br />
<br />
<br />
One possible purchaser will be the Cancer Research Technology Limited (CRT). It is the cancer-focused technology development and commercialisation arm of Cancer Research UK, the world’s largest cancer charity. Their aim is providing best in class technology transfer services to cancer researchers. The ProteoLux®system will complete their platform technology and research tools.<br />
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<font size="1">This list is far from being exhaustive.</font><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancerTeam:ESBS-Strasbourg/proteolux/application/cancer2010-11-30T10:25:47Z<p>Georgio: </p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
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<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">Neurodegenerative Diseases</div><br />
<div class="desc"><br />
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<ul><br />
<li><a href="#alzeimer">Alzeimer disease</a></li><br />
<li><a href="#costs">Occurrence and costs</a></li><br />
<li><a href="#genetics">Genetics</a></li><br />
<li><a href="#pathology">Pathology</a></li><br />
<li><a href="#tau">The Tau protein</a></li><br />
<li><a href="#calpainss">Calpains</a></li><br />
<li><a href="#caspases">Caspases</a></li><br />
<li><a href="#proteolux">Utilization of the ProteoLux® Pro system</a></li><br />
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<a name="cancer"></a><br />
ProteoLux® Pro system against cancer<br />
</div><br />
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<b>Proteomics</b> technology is an approach of science to understand the expression of the whole set of proteins and its function at the cellular level. Its role in cancer research becomes more significant nowadays. Consulting Medline reveals that, since 1997, when the word proteomics first appeared as an entry, approx. 0.12% of all articles on cancer also contain the word proteomics. Comparing this frequency with the occurrence of the search string ‘genomics’ in 0.17% of the cancer literature, it becomes obvious that proteomics has attracted major scientific attention in the field of cancer research. Vice versa, a search for ‘cancer AND proteomics’ shows that 16% of the proteomics literature deals with cancer, testifying that the attraction is mutual. <br />
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Indeed, proteomics technology enables the researchers to look into the proteins level of the cancer cells, proteins being the physiological and pathological indispensible players. Proteins represent the majority of drug targets. However, currently only around 500 out of the estimated >3000 proteins that are predicted to be druggable are exploited.<br />
<br><br><br />
<b>Proteomics has the great potential to be one of the most powerful tools for cancer research.</b> The <b>ProteoLux® Pro system offers a new proteomic tool for cancer research.</b> It can help in therapy designing by characterizing the potency of drug targets. The multiple targets of the system and its advantages will be exemplified in the case of the pancreatic ductal adenocarcinoma (PDAC), a major unsolved health problem.<br />
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Pancreatic ductal adenocarcinoma (PDAC)<br />
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Pancreas is a gland organ found in vertebrates. The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. It presents endocrine and exocrine functions. In fact this gland permits to produce hormones like insulin and to secrete digestive enzymes contained in a pancreatic juice which are inserted into the small intestine to help for assimilation of nutrients. The exocrine pancreas consists of acinar and duct cells. The Figure 12 describes in detail the pancreas anatomy. <br />
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<font size="1">Figure 1 | Anatomy of the pancreas.<br><br />
The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. The exocrine pancreas consists of acinar and duct cells. The acinar cells produce digestive enzymes and constitute the bulk of the pancreatic tissue. They are organized into grape-like clusters that are at the smallest termini of the branching duct system. The ducts, which add mucous and bicarbonate to the enzyme mixture, form a network of increasing size, culminating in main and accessory pancreatic ducts that empty into the duodenum. The endocrine pancreas, consisting of four specialized cell types that are organized into compact islets embedded within acinar tissue, secretes hormones into the bloodstream. The α- and β-cells regulate the usage of glucose through the production of glucagon and insulin, respectively. Pancreatic polypeptide and somatostatin that are produced in the PP and δ-cells modulate the secretory properties of the other pancreatic cell types. a | Gross anatomy of the pancreas. b | The exocrine pancreas. c | A single acinus. d | A pancreatic islet embedded in exocrine tissue.<br><br />
Bardeesy N and DePinho R A. (2002) ”PANCREATIC CANCER BIOLOGY AND GENETICS” Nature Reviews Cancer VOL.2 pp897-909<br />
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Pancreatic Cancer is a cancer of the digestive system. Different kinds of pancreatic cancer exist : Adenocarcinomas, which represents 95% of cases, adenosquamous carcinomas, singet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas and undifferentiated carcinomas with osteoclast-like giant cells which represent the remaining 5%. <br />
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<font size="1">Figure 2 | Gross photograph of an infiltrating adenocarcinoma<br><br />
Note the dramatic narrowing of the pancreatic duct associated with the poorly defined white neoplasm.<br><br />
Maitra A, 1,2 and Ralph H. Hruban R H. (2008) “Pancreatic cancer” Annu. Rev. Pathol. Mech. Dis. 3:157–88<br />
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<b>Pancreatic ductal adenocarcinoma</b> is a malignant tumor of epithelia originating in glandular tissue. It grossly produces a firm, highly sclerotic mass (Figure 2). <b>Aggressive and devastating disease</b>, it is one of the most common causes of cancer related death. It is characterized by invasiveness, rapid progression and <b>profound resistance to treatment.</b><br />
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<font size="1">Figure 3 | Mortality of the pancreas cancer per year<br><br />
<a href="http://commons.wikimedia.org/wiki/File:Pancreas_cancer_world_map_-_Death_-_WHO2004.svg">link</a><br />
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Pancreatic cancer is predominantly a disease of the elderly. Besides, men are more concerned than women. Age and gender are so risk factors. Other established risk factors include diets high in meats and fat, low serum folate levels, obesity, long-standing diabetes mellitus, and chronic pancreatitis. Pancreatic cancer may be genetic. This is the case for 5 to 10% of patient but the gene responsible of this disease is not yet identified. Furthermore for the moment there are no real guidelines for preventing from pancreatic cancer.<br />
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Developed countries are the most affected by this disease as shown in Figure 3. <b>This cancer causes the death of thousands people in the world each year</b> of whom 7000 French and 36 800 American.<br />
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This cancer is <b>very hard to detect.</b> Indeed, there is often no symptom attached to early pancreatic cancer. Furthermore the later symptoms are non specific and varied. That is why this disease is also called “silent killer”. Pain in the abdomen, loss of appetite and so loss of weight, painless jaundice, diabetes mellitus or elevated blood sugar level, trousseau sign or clinical depression are common symptoms of pancreatic cancer. <br />
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The <b>prognosis</b> of the pancreatic cancer is poor. The <b>5- year survival rate</b> of all patients is <b>below 5%</b>, and the median survival time after diagnosis is 6 months. <b>Surgery offered the only possibility of cure.</b> But surgery may only be used if the tumor is not too advanced and if the cancer does not present metastasis. This surgery is not easy because of the high amount of veins surrounding the pancreas and this operation is possible only in 20% of cases. Furthermore a relapse is common (70 to 80% of people). Thus, only 20% of the patients who have undergone surgery survive longer than 5 years. For those who cannot undergo this surgery their median survey is approximately of 6 months. Radiotherapy may be used in case of evolved cancer and is established after surgery. Then chemotherapy may be used when the cancer presents metastasis. Gemcitabine, Fluorouracil, cisplatine and oxaliplatine are used. Nevertheless, <b>PDAC is insensitive to most therapies including chemotherapy, radiotherapy and immunotherapy.</b><br />
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Molecular Pathogenesis of PDAC<br />
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<font size="1">Figure 4 |Progression model of pancreatic adenocarcinoma<br><br />
<a href="http://www.nature.com/modpathol/journal/v15/n4/images/3880544f1.jpg + medscape.com">link</a><br />
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The progression from histologically normal epithelium to low-grade pancreatic intraepithelial neoplasia (PanINs are microscopic lesions in the smaller (<5 mm) pancreatic ducts), to high-grade PanIN, to invasive carcinoma (left to right in Figure 4) is associated with the accumulation of specific genetic alterations. This signature molecular profile consists notably in <b>mutations in the oncogene K-RAS and the tumor suppressors CDKN2, TP53, SMAD4/DPC4 and BRCA2.</b> Other recognized precursor lesions of adenocarcinoma (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) likely harbor a distinct compendium of genetic alterations in their path to invasive cancer. <br />
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The Figure 5 presents a schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma.<br />
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<img src="https://static.igem.org/mediawiki/2010/8/8b/ESBS-Strasbourg-Pancreas5.png" width="400px" height="310px"></a><br />
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<div style="position: relative; width: 400px; height: 150px; id="layer1" align="justify"><br />
<font size="1">Figure 5 | Schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma<br><br />
O'Reilly E and Epstein A (2010) “Recent Findings in Pancreatic Cancer: Illuminating Emerging Treatment Strategies” MedscapeCME Oncology <br><br><br />
<br />
EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinases; FGF-R = fibroblast growth factor receptor; GDP = guanosine diphosphate; GTP = guanosine triphosphate; HER2 = human EGFR 2; IGF = insulin growth factor; MEK = mitogen-activated protein kinase; MEKK = mitogen-activated protein kinase kinase; MMP = matrix metalloproteinase; mTOR = mammalian target of rapamycin; NF-kappaB = nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF = vascular endothelial growth factor receptor. <br />
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The <b>majority (90%) of pancreatic adenocarcinomas</b> have a <b>mutated K-Ras oncogene.</b> KRAS encodes a member of the RAS family of guanosine triphosphate (GTP)-binding proteins that mediate a range of cellular functions, including proliferation, cell survival, cytoskeletal remodeling, and motility, among others. Activating mutations impair the intrinsic GTPase activity of the KRAS gene product, resulting in a protein that is constitutively active in intracellular signal transduction. In addition to its role in pancreatic cancer initiation, constitutive RAS signaling appears to be required for pancreatic cancer maintenance as well.<br />
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For many years targeting, Ras has been a holy grail in the treatment of pancreatic cancer. Either single-agent or combination therapies with farnesyl transferase inhibitors (SWOG 9924 study) that target Ras translocation have shown limited activity. <b>Ras</b> still represents an <b>attractive and ubiquitous target.</b><br />
<br><br><br />
Activated Kras engages a number of downstream effector pathways, including RAF–mitogen-activated protein kinase (RAFMAPK), phosphoinositide-3-kinase (PI3K), and RalGDS pathways (as shown in Figure 6) producing thus a remarkable array of cellular effects, including induction of proliferation, survival and invasion through the stimulation of several effector pathways. <br />
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<center><br />
<img src="https://static.igem.org/mediawiki/2010/3/39/ESBS-Strasbourg-Pancreas6.png" width="400px" height="292px"></a><br />
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<center><br />
<div style="position: relative; width: 400px; height: 40px; id="layer1" align="justify"><br />
<font size="1">Figure 6 | Some pathways in which KRAS is involved in PDAC<br><br />
Lauth M, Toftgard R and al (2010) “DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS” Nat. Struct. Mol. Biol. 17, 718 - 725<br />
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<p><b>Mirk/Dyrk1B as potential target in pancreas cancer </b></p><br />
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<b>DYRK1B/Mirk</b> is a RAS effector kinase (Figure 6), which is activated by phosphorylation by its upstream activator, the MAP kinase kinase MKK3, suggesting that Mirk, like p38, is activated by certain environmental stress agents. <br />
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Mirk/Dyrk1B is a member of the Minibrain/dyrk family of kinases, more precisely an arginine-directed serine/threonine protein kinase. It is expressed at low levels in most normal tissues but at elevated levels in quiescent pancreatic cancer cells. Mirk is expressed in about 90% of pancreatic cancers and is amplified in a subset. Mirk appears not to be an essential gene for normal cells from embryonic knockout studies in mice and RNA interference studies on cultured cells. Mirk/Dyrk1B mediates the prolonged survival of cancer cells through increasing expression of a cohort of antioxidant genes. These unusual characteristics suggest that Mirk may be a selective target for therapeutic intervention [70].<br />
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Indeed, one major issue in pancreas cancer is the insensitivity to treatment due to the activation of defense systems, notably those producing antioxydants in which Mirk acts. By inhibiting Mirk, the protection of cancer cells will be inhibited, making them more sensitive to actual treatment (chemotherapy, radiotherapy…). <br />
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<p><b>Advantages of the ProteoLux® Pro system in cancer research</b></p><br />
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The <b>ProteoLux® Pro system enable to</b> <font color="#800000"><b>degrade specifically one or more proteins expressed in vivo at a given time and in a given cell type.</b></font><br />
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The <b>ProteoLux® Pro system also enables you to control the concentration of a protein according to the lighting alternation between far red light and red light. The ProteoLux® Pro system also enables you to degrade specifically muted protein (not the normal one) by using specific intrabodies.</b><br />
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<br />
<p><b><u>The ProteoLux® Pro system : an amazing tool to study every overexpressed protein, notably oncoproteins.</u></b></p><br />
<br><br />
Indeed, oncogenes which code for oncoproteins are mutated proto-oncogenes, very important genes involved in the regulation of cell cycle, cell growth and cell proliferation. Mutations in proto-oncogenes can lead:<br />
<br><br><br />
<ul><br />
<li>either to a conformational change of the oncoprotein, thus to an increase of its activity or a loss of its regulation</li><br />
<li>or to an increase of its activity, an increase of its stability, a duplication of the oncogene, a retroviral insertion, all this leading to an increase of the concentration of the oncoprotein</li><br />
<li>or to a chromosomic translocation (expression in the wrong cell type, at the wrong time or constitutive expression of the oncogene).</li><br />
</ul><br />
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<p><b><u>The ProteoLux® Pro system : a rapid access to the potency of therapeutic targets</u></b></p><br />
<br><br />
The potency of Mirk as a therapeutic target can easily and relatively fastly be studied with the ProteoLux® system.<br />
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<div style="position: relative; width: 400px; height: 30px; id="layer1" align="justify"><br />
<font size="1">Figure 7 | Principle of the assessment of the potency as therapeutic target by the ProteoLux® Pro system<br />
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Besides, by using several intrabodies it is possible to target several proteins at the same time. Therefore, the ProteoLux® Pro system permits an easy assessment of combinatory targeting.<br />
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<p><b><u>The ProteoLux® Pro system : solution to the specific difficulty of clinical management in cancer</u></b></p><br />
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<br />
As shown in Figure 4, tumors possess cytogenetically different clones corresponding to different stages of progression of the disease. This heterogeneity contributes to differences in clinical behavior and responses to treatment of tumors of the same diagnostic type. With the ProteoLux® system you can target several muted proteins by intrabodies. Thus, you will only target muted proteins but all the muted proteins. Therefore you will target all the stages of progression of the disease.<br />
<br><br><br />
The discovery and understanding of the altered genes causing cancer (oncogenes, tumor-suppressor genes, and microRNA genes) and of the pathways involved permit the development of cancer therapies. Unfortunately, research goes slowly. The <b>ProteoLux® Pro system gives research a boost.</b><br />
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<div class="heading"><br />
<a name="purchaser"></a><br />
Our potential purchaser<br />
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The ProteoLux® system can interest big pharmaceutical firms like Roche or Novartis which are already focusing on pancreatic cancer. Indeed, Roche has developed a molecule for chemotherapy, Traceva and Novartis has created a new product Afinitor, which permit also to increase life expectancy of people affect by pancreatic endocrine tumor. Our system permits these companies to broaden their scope against pancreatic cancer by studying easily the potency of a new target. Notably, they will be able to develop cancer stage-specific drugs basing on the ProteoLux® system.<br />
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One possible purchaser will be the Cancer Research Technology Limited (CRT). It is the cancer-focused technology development and commercialisation arm of Cancer Research UK, the world’s largest cancer charity. Their aim is providing best in class technology transfer services to cancer researchers. The ProteoLux®system will complete their platform technology and research tools.<br />
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<font size="1">This list is far from being exhaustive.</font><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancerTeam:ESBS-Strasbourg/proteolux/application/cancer2010-11-30T10:16:40Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
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<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">Neurodegenerative Diseases</div><br />
<div class="desc"><br />
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<ul><br />
<li><a href="#alzeimer">Alzeimer disease</a></li><br />
<li><a href="#costs">Occurrence and costs</a></li><br />
<li><a href="#genetics">Genetics</a></li><br />
<li><a href="#pathology">Pathology</a></li><br />
<li><a href="#tau">The Tau protein</a></li><br />
<li><a href="#calpainss">Calpains</a></li><br />
<li><a href="#caspases">Caspases</a></li><br />
<li><a href="#proteolux">Utilization of the ProteoLux® Pro system</a></li><br />
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ProteoLux® Pro system against cancer<br />
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<b>Proteomics</b> technology is an approach of science to understand the expression of the whole set of proteins and its function at the cellular level. Its role in cancer research becomes more significant nowadays. Consulting Medline reveals that, since 1997, when the word proteomics first appeared as an entry, approx. 0.12% of all articles on cancer also contain the word proteomics. Comparing this frequency with the occurrence of the search string ‘genomics’ in 0.17% of the cancer literature, it becomes obvious that proteomics has attracted major scientific attention in the field of cancer research. Vice versa, a search for ‘cancer AND proteomics’ shows that 16% of the proteomics literature deals with cancer, testifying that the attraction is mutual. <br />
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Indeed, proteomics technology enables the researchers to look into the proteins level of the cancer cells, proteins being the physiological and pathological indispensible players. Proteins represent the majority of drug targets. However, currently only around 500 out of the estimated >3000 proteins that are predicted to be druggable are exploited.<br />
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<b>Proteomics has the great potential to be one of the most powerful tools for cancer research.</b> The <b>ProteoLux® Pro system offers a new proteomic tool for cancer research.</b> It can help in therapy designing by characterizing the potency of drug targets. The multiple targets of the system and its advantages will be exemplified in the case of the pancreatic ductal adenocarcinoma (PDAC), a major unsolved health problem.<br />
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Pancreatic ductal adenocarcinoma (PDAC)<br />
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Pancreas is a gland organ found in vertebrates. The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. It presents endocrine and exocrine functions. In fact this gland permits to produce hormones like insulin and to secrete digestive enzymes contained in a pancreatic juice which are inserted into the small intestine to help for assimilation of nutrients. The exocrine pancreas consists of acinar and duct cells. The Figure 12 describes in detail the pancreas anatomy. <br />
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<font size="1">Figure 1 | Anatomy of the pancreas.<br><br />
The pancreas is comprised of separate functional units that regulate two major physiological processes: digestion and glucose metabolism. The exocrine pancreas consists of acinar and duct cells. The acinar cells produce digestive enzymes and constitute the bulk of the pancreatic tissue. They are organized into grape-like clusters that are at the smallest termini of the branching duct system. The ducts, which add mucous and bicarbonate to the enzyme mixture, form a network of increasing size, culminating in main and accessory pancreatic ducts that empty into the duodenum. The endocrine pancreas, consisting of four specialized cell types that are organized into compact islets embedded within acinar tissue, secretes hormones into the bloodstream. The α- and β-cells regulate the usage of glucose through the production of glucagon and insulin, respectively. Pancreatic polypeptide and somatostatin that are produced in the PP and δ-cells modulate the secretory properties of the other pancreatic cell types. a | Gross anatomy of the pancreas. b | The exocrine pancreas. c | A single acinus. d | A pancreatic islet embedded in exocrine tissue.<br><br />
Bardeesy N and DePinho R A. (2002) ”PANCREATIC CANCER BIOLOGY AND GENETICS” Nature Reviews Cancer VOL.2 pp897-909<br />
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Pancreatic Cancer is a cancer of the digestive system. Different kinds of pancreatic cancer exist : Adenocarcinomas, which represents 95% of cases, adenosquamous carcinomas, singet ring cell carcinomas, hepatoid carcinomas, colloid carcinomas, undifferentiated carcinomas and undifferentiated carcinomas with osteoclast-like giant cells which represent the remaining 5%. <br />
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<font size="1">Figure 2 | Gross photograph of an infiltrating adenocarcinoma<br><br />
Note the dramatic narrowing of the pancreatic duct associated with the poorly defined white neoplasm.<br><br />
Maitra A, 1,2 and Ralph H. Hruban R H. (2008) “Pancreatic cancer” Annu. Rev. Pathol. Mech. Dis. 3:157–88<br />
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<b>Pancreatic ductal adenocarcinoma</b> is a malignant tumor of epithelia originating in glandular tissue. It grossly produces a firm, highly sclerotic mass (Figure 2). <b>Aggressive and devastating disease</b>, it is one of the most common causes of cancer related death. It is characterized by invasiveness, rapid progression and <b>profound resistance to treatment.</b><br />
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<font size="1">Figure 3 | Mortality of the pancreas cancer per year<br><br />
http://commons.wikimedia.org/wiki/File:Pancreas_cancer_world_map_-_Death_-_WHO2004.svg<br />
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Pancreatic cancer is predominantly a disease of the elderly. Besides, men are more concerned than women. Age and gender are so risk factors. Other established risk factors include diets high in meats and fat, low serum folate levels, obesity, long-standing diabetes mellitus, and chronic pancreatitis. Pancreatic cancer may be genetic. This is the case for 5 to 10% of patient but the gene responsible of this disease is not yet identified. Furthermore for the moment there are no real guidelines for preventing from pancreatic cancer.<br />
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Developed countries are the most affected by this disease as shown in Figure 3. <b>This cancer causes the death of thousands people in the world each year</b> of whom 7000 French and 36 800 American.<br />
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This cancer is <b>very hard to detect.</b> Indeed, there is often no symptom attached to early pancreatic cancer. Furthermore the later symptoms are non specific and varied. That is why this disease is also called “silent killer”. Pain in the abdomen, loss of appetite and so loss of weight, painless jaundice, diabetes mellitus or elevated blood sugar level, trousseau sign or clinical depression are common symptoms of pancreatic cancer. <br />
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The <b>prognosis</b> of the pancreatic cancer is poor. The <b>5- year survival rate</b> of all patients is <b>below 5%</b>, and the median survival time after diagnosis is 6 months. <b>Surgery offered the only possibility of cure.</b> But surgery may only be used if the tumor is not too advanced and if the cancer does not present metastasis. This surgery is not easy because of the high amount of veins surrounding the pancreas and this operation is possible only in 20% of cases. Furthermore a relapse is common (70 to 80% of people). Thus, only 20% of the patients who have undergone surgery survive longer than 5 years. For those who cannot undergo this surgery their median survey is approximately of 6 months. Radiotherapy may be used in case of evolved cancer and is established after surgery. Then chemotherapy may be used when the cancer presents metastasis. Gemcitabine, Fluorouracil, cisplatine and oxaliplatine are used. Nevertheless, <b>PDAC is insensitive to most therapies including chemotherapy, radiotherapy and immunotherapy.</b><br />
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Molecular Pathogenesis of PDAC<br />
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<font size="1">Figure 4 |Progression model of pancreatic adenocarcinoma<br><br />
http://www.nature.com/modpathol/journal/v15/n4/images/3880544f1.jpg + medscape.com<br />
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The progression from histologically normal epithelium to low-grade pancreatic intraepithelial neoplasia (PanINs are microscopic lesions in the smaller (<5 mm) pancreatic ducts), to high-grade PanIN, to invasive carcinoma (left to right in Figure 4) is associated with the accumulation of specific genetic alterations. This signature molecular profile consists notably in <b>mutations in the oncogene K-RAS and the tumor suppressors CDKN2, TP53, SMAD4/DPC4 and BRCA2.</b> Other recognized precursor lesions of adenocarcinoma (intraductal papillary mucinous neoplasm and mucinous cystic neoplasm) likely harbor a distinct compendium of genetic alterations in their path to invasive cancer. <br />
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The Figure 5 presents a schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma.<br />
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<font size="1">Figure 5 | Schematic diagram of intracellular signalling pathways in pancreatic adenocarcinoma<br><br />
O'Reilly E and Epstein A (2010) “Recent Findings in Pancreatic Cancer: Illuminating Emerging Treatment Strategies” MedscapeCME Oncology <br><br><br />
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EGFR = epidermal growth factor receptor; ERK = extracellular signal-regulated kinases; FGF-R = fibroblast growth factor receptor; GDP = guanosine diphosphate; GTP = guanosine triphosphate; HER2 = human EGFR 2; IGF = insulin growth factor; MEK = mitogen-activated protein kinase; MEKK = mitogen-activated protein kinase kinase; MMP = matrix metalloproteinase; mTOR = mammalian target of rapamycin; NF-kappaB = nuclear factor kappa-light-chain-enhancer of activated B cells; VEGF = vascular endothelial growth factor receptor. <br />
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The <b>majority (90%) of pancreatic adenocarcinomas</b> have a <b>mutated K-Ras oncogene.</b> KRAS encodes a member of the RAS family of guanosine triphosphate (GTP)-binding proteins that mediate a range of cellular functions, including proliferation, cell survival, cytoskeletal remodeling, and motility, among others. Activating mutations impair the intrinsic GTPase activity of the KRAS gene product, resulting in a protein that is constitutively active in intracellular signal transduction. In addition to its role in pancreatic cancer initiation, constitutive RAS signaling appears to be required for pancreatic cancer maintenance as well.<br />
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For many years targeting, Ras has been a holy grail in the treatment of pancreatic cancer. Either single-agent or combination therapies with farnesyl transferase inhibitors (SWOG 9924 study) that target Ras translocation have shown limited activity. <b>Ras</b> still represents an <b>attractive and ubiquitous target.</b><br />
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Activated Kras engages a number of downstream effector pathways, including RAF–mitogen-activated protein kinase (RAFMAPK), phosphoinositide-3-kinase (PI3K), and RalGDS pathways (as shown in Figure 6) producing thus a remarkable array of cellular effects, including induction of proliferation, survival and invasion through the stimulation of several effector pathways. <br />
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<font size="1">Figure 6 | Some pathways in which KRAS is involved in PDAC<br><br />
Lauth M, Toftgard R and al (2010) “DYRK1B-dependent autocrine-to-paracrine shift of Hedgehog signaling by mutant RAS” Nat. Struct. Mol. Biol. 17, 718 - 725<br />
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<p><b>Mirk/Dyrk1B as potential target in pancreas cancer </b></p><br />
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<b>DYRK1B/Mirk</b> is a RAS effector kinase (Figure 6), which is activated by phosphorylation by its upstream activator, the MAP kinase kinase MKK3, suggesting that Mirk, like p38, is activated by certain environmental stress agents. <br />
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Mirk/Dyrk1B is a member of the Minibrain/dyrk family of kinases, more precisely an arginine-directed serine/threonine protein kinase. It is expressed at low levels in most normal tissues but at elevated levels in quiescent pancreatic cancer cells. Mirk is expressed in about 90% of pancreatic cancers and is amplified in a subset. Mirk appears not to be an essential gene for normal cells from embryonic knockout studies in mice and RNA interference studies on cultured cells. Mirk/Dyrk1B mediates the prolonged survival of cancer cells through increasing expression of a cohort of antioxidant genes. These unusual characteristics suggest that Mirk may be a selective target for therapeutic intervention [70].<br />
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Indeed, one major issue in pancreas cancer is the insensitivity to treatment due to the activation of defense systems, notably those producing antioxydants in which Mirk acts. By inhibiting Mirk, the protection of cancer cells will be inhibited, making them more sensitive to actual treatment (chemotherapy, radiotherapy…). <br />
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<p><b>Advantages of the ProteoLux® Pro system in cancer research</b></p><br />
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The <b>ProteoLux® Pro system enable to</b> <font color="#800000"><b>degrade specifically one or more proteins expressed in vivo at a given time and in a given cell type.</b></font><br />
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The <b>ProteoLux® Pro system also enables you to control the concentration of a protein according to the lighting alternation between far red light and red light. The ProteoLux® Pro system also enables you to degrade specifically muted protein (not the normal one) by using specific intrabodies.</b><br />
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<p><b><u>The ProteoLux® Pro system : an amazing tool to study every overexpressed protein, notably oncoproteins.</u></b></p><br />
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Indeed, oncogenes which code for oncoproteins are mutated proto-oncogenes, very important genes involved in the regulation of cell cycle, cell growth and cell proliferation. Mutations in proto-oncogenes can lead:<br />
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<li>either to a conformational change of the oncoprotein, thus to an increase of its activity or a loss of its regulation</li><br />
<li>or to an increase of its activity, an increase of its stability, a duplication of the oncogene, a retroviral insertion, all this leading to an increase of the concentration of the oncoprotein</li><br />
<li>or to a chromosomic translocation (expression in the wrong cell type, at the wrong time or constitutive expression of the oncogene).</li><br />
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<p><b><u>The ProteoLux® Pro system : a rapid access to the potency of therapeutic targets</u></b></p><br />
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The potency of Mirk as a therapeutic target can easily and relatively fastly be studied with the ProteoLux® system.<br />
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<font size="1">Figure 7 | Principle of the assessment of the potency as therapeutic target by the ProteoLux® Pro system<br />
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Besides, by using several intrabodies it is possible to target several proteins at the same time. Therefore, the ProteoLux® Pro system permits an easy assessment of combinatory targeting.<br />
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<p><b><u>The ProteoLux® Pro system : solution to the specific difficulty of clinical management in cancer</u></b></p><br />
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As shown in Figure 4, tumors possess cytogenetically different clones corresponding to different stages of progression of the disease. This heterogeneity contributes to differences in clinical behavior and responses to treatment of tumors of the same diagnostic type. With the ProteoLux® system you can target several muted proteins by intrabodies. Thus, you will only target muted proteins but all the muted proteins. Therefore you will target all the stages of progression of the disease.<br />
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The discovery and understanding of the altered genes causing cancer (oncogenes, tumor-suppressor genes, and microRNA genes) and of the pathways involved permit the development of cancer therapies. Unfortunately, research goes slowly. The <b>ProteoLux® Pro system gives research a boost.</b><br />
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<a name="purchaser"></a><br />
Our potential purchaser<br />
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The ProteoLux® system can interest big pharmaceutical firms like Roche or Novartis which are already focusing on pancreatic cancer. Indeed, Roche has developed a molecule for chemotherapy, Traceva and Novartis has created a new product Afinitor, which permit also to increase life expectancy of people affect by pancreatic endocrine tumor. Our system permits these companies to broaden their scope against pancreatic cancer by studying easily the potency of a new target. Notably, they will be able to develop cancer stage-specific drugs basing on the ProteoLux® system.<br />
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One possible purchaser will be the Cancer Research Technology Limited (CRT). It is the cancer-focused technology development and commercialisation arm of Cancer Research UK, the world’s largest cancer charity. Their aim is providing best in class technology transfer services to cancer researchers. The ProteoLux®system will complete their platform technology and research tools.<br />
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<font size="2">This list is far from being exhaustive.</font><br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Pancreas7.pngFile:ESBS-Strasbourg-Pancreas7.png2010-11-30T10:07:45Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Pancreas6.pngFile:ESBS-Strasbourg-Pancreas6.png2010-11-30T09:52:35Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Pancreas5.pngFile:ESBS-Strasbourg-Pancreas5.png2010-11-30T09:44:53Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Pancreas4.pngFile:ESBS-Strasbourg-Pancreas4.png2010-11-30T09:32:12Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Pancreas3.pngFile:ESBS-Strasbourg-Pancreas3.png2010-11-30T09:22:15Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-pancreas2.pngFile:ESBS-Strasbourg-pancreas2.png2010-11-30T09:09:47Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-pancreas1.pngFile:ESBS-Strasbourg-pancreas1.png2010-11-30T09:00:16Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuroTeam:ESBS-Strasbourg/proteolux/application/neuro2010-11-30T08:53:37Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<div class="heading">Neurodegenerative Diseases</div><br />
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<ul><br />
<li><a href="#alzeimer">Alzeimer disease</a></li><br />
<li><a href="#costs">Occurrence and costs</a></li><br />
<li><a href="#genetics">Genetics</a></li><br />
<li><a href="#pathology">Pathology</a></li><br />
<li><a href="#tau">The Tau protein</a></li><br />
<li><a href="#calpainss">Calpains</a></li><br />
<li><a href="#caspases">Caspases</a></li><br />
<li><a href="#proteolux">Utilization of the ProteoLux® Pro system</a></li><br />
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<a name="alzeimer"></a><br />
ProteoLux Pro ® against the Alzheimer disease<br />
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<p><b>Alzeimer disease</b><p><br />
<br><br />
The Alzheimer disease (AD) is a common form of dementia. This neurodegenerative and terminal disease was first described by German neuropathologist Alois Alzheimer in 1906 and was named after him. Its most prevalent form occurs by person over 65 years and it is responsible for ca. 60 percent of the 24 million dementia cases [54]. <br />
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Characteristic for the disease is a deterioration of cognitive capacities, which follows by a decrease of regular activities and apparent behavioral disorders. <br />
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<a name="costs"></a><br />
Occurrence and costs<br />
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Alzheimer occurs nearly exclusively by people of high age. A rising prevalent can be observed in countries with an aging population. By people under 65 of age ca. 2% are affected, by 70 year old people it is nearly 3%, by the 75 years old 6% and by the 85 year old 20% of the people are affected [54]. <br />
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In 2007, 29 million people worldwide were affected by the AD. A computer simulation based on the population date of the United Nations showed that 106 million people will be affected by AD in 2050 [55]. <br />
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Dementias like Alzheimer are one of the most costly diseases in the United States and Europe and can cause massive problems today and in the future. The costs are direct medical costs such as nursing home care or nonmedical cost such as day care with a loss of productivity of the caregiver. The worldwide costs of dementia have been estimated to 160 billion dollar per year. They will dramatically increase in coming years. [56]<br />
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Genetics<br />
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There exists a genetic component which causes AD. Five to ten percent of the affected persons show a familiar accumulation or FAD (familial Alzheimer Disease) which results from mutations of the presenilin 1 gene on the chromosome 14, on the presenilin 2 gene on chromosome 1 or the APP (Amyloid-Precursor-Protein) gene on chromosome 21 [57]. <br />
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The Down syndrome with tripled chromosome 21 also heightens the risk of developing dementia like Alzheimer, but a diagnosis is often difficult due to already affected cognitive capacities of these patients. <br />
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Also a mutation variant of the SORL1 gene is known as a risk factor which heightens change of becoming affected by the Alzheimer disease. [58]<br />
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Pathology<br />
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By patients affected senile plaques and fibril accretion are formed. The protein accretions of the plaques consist mostly of beta amyloid peptide. The intracellular located neurofibrils consist of Tau protein. This protein aggregates to fibrils when it is hyper phosphorylated. It is not yet clear if this phosphorylation is of secondary nature or if its triggers the disease. [59]<br />
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During the cause of disease the brain mass decreases due to a dyeing of neurons which is called a Brain atrophy. Additionally the neurotransmitter acetylcholine is not produced in sufficient quantity. The reason for this diminished production is an under expression of the enzyme cholinacetyltranferase which catalyzes the reaction of acetyl CoA and choline which leads to a general decrease of congenital capacities. [59] <br />
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The precursor of the Aβ peptide is the Amyloid precursor protein (APP), which is an integral membrane protein. Most of the protein protrudes out of the cell into the extracellular matrix, while just a small part is in the intracellular space of the cell. It is a type one trans membrane protein by which the amino terminus is outside and the carboxyl terminus is inside the cell. [59]<br />
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APP is cleaved by certain different secretases (alpha-, beta-, and gamma-secretase) which can result in a release of the Aβ peptide out of the precursor protein. There are basically two different mechanism existing. [59]<br />
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<ol><br />
<li>The amyloidogenic mechanism : APP is first cleaved by the beta secretase and then by the gamma secretase. This incision within the transmembrane domain leads to a release of the Aβ peptide. </li><br />
<li>The non amyloidogenic mechanism: APP is cleaved by an alpha- secretase. The incision takes place within a part of the APP which contains Aβ. This prevents from the generation of the Aβ peptide. The result is a release of a relatively big part of the protein which further mechanism is not clear yet.</li><br />
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<font size="1">Figure 1 | The two different mechanisms of APP cleavage<br><br />
By the non amyloidogenic mechanism APP is cleaved by an alpha secretase (ADAM 9,10 or 17) with the extracellular domain. In the amyloidogenic mechanism APP is first cleaved by a beta secretase (BACE1) and followed by a second incision of a gamma secretase (PEN-2, APH-1 and Nicastrin) which releases the Aβ peptide<br />
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Both pathways can take place parallel in neuron cells. The length of the Aβ peptides can vary. The main type is the Aβ 40 peptide consisting of 40 amino acids, Aβ 42 peptide being represented in a smaller part. The length of the peptide is significant as the longer peptide with 42 amino acids has a far higher tendency for aggregation as the smaller peptide. [59]<br />
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Another factor for the Alzheimer disease is a dysfunction of mitochondrion. A blockade of the respiratory chain at the complex IV leads to a high production of radicals which can damage the cells. If this blockade is a result of the high Aβ production or if Aβ is produced as an antioxidant to deal with the high oxidative stress is not yet clear. <br />
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<a name="tau"></a><br />
The Tau protein<br />
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The function of the tau protein is to sustain the stability of the cytoskeleton and the proteins which are attached by the microtubules. The Tau protein is regulated by the MAPT gene where a mutation can lead to several diseases like pick diseases or corticobasal degeneration. In the Alzheimer disease accretion of Tau protein leads to senile plaques. In theory it is believed that these plaques disrupt the calcium homeostasis of the cells leading to an apoptosis. [60]<br />
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Posttranslational alterations on the MAPT gene on chromosome 17 result in nine isoforms of the gene in the human central nervous system. By the construction of filament structures single Tau proteins are joined over their repeating part. The amino terminal end is then cleaved. A hyper phosphorylation of paired helical Tau filaments leads to a misfolded not functional protein which is unable to interact with microtubules. As a result the accretion of these neurofibrillary lesions can be observed in the brains of Alzheimer patients [61]. <br />
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As demonstrated previously, the Tau protein and its fragments play a very important role in the Alzheimer disease. Indeed, it has been shown that truncated Tau protein was present in Alzheimer disease and that the cleavage of tau is an early event of the Alzheimer disease development. The cleavage of Tau into fragments is done by several proteases such as caspases and calpains. However the real implication of the Tau protein as well as its level of degradation and aggregation is not fully understood yet. Some studies have been conducted on the Tau fragments, the proteases causing those fragments and the aggregation of those resulting fragments. So far, even though those aggregates have been shown to be involved in Alzheimer diseases, there has not been any clear and conclusive result allowing the establishment of the importance and the role of those aggregates. <br />
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The <b>ProteoLux® Pro system</b> can be used to <b>help finding a new therapeutical target against the calpains</b> as the research in this domain is quite advanced and the use of a tightly controlled system can improve the existing knowledge. On the other hand, the ProteoLux® system can be <b>used in fundamental research to gather datas on the caspases and their real involvement in the Alzheimer disease.</b> It has been clearly shown that caspases might have a role in the degradation of Tau protein into fragments. However there is no clear result pointing out their exact implication and the importance of this implication compared to the calpains. Therefore more datas are first required before going more into details with the caspase research as pharmacological target. [62]<br />
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<a name="calpains"></a><br />
Calpains<br />
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Calpains are nonlysosomal, neutral cysteine proteases activated by calcium. There are two types of calpains expressed in the brain, the most characterized among the calpains. They are the µ-calpain and the m-calpain. They are presented as a heterodimer form. Until now the difference in activity between those two forms of calpains is not known, because they also have the same substrate. Normal transient activation of calpains in a normal functioning cell is involved in cell signaling, synaptic plasticity, and protein turnover. In contrast, sustained activation of calpains contributes to both chronic neurodegeneration associated with Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. <br />
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Several calpains inhibitors already exist for the study of the calpains activity, however they do not allow a tight control of the calpain concentration or activity in the cell. <b>That is where the ProteoLux® Pro system comes into action. </b><br />
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<a name="caspases"></a><br />
Caspases<br />
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As said previously, the caspases’ role in the Alzheimer disease has not been clearly defined yet. <b>The ProteoLux® Pro system could be a major tool in the study of this caspase system and the discovery of new leading knowledge on the caspases.</b> All the new knowledge acquired could be further developed to reach a level such as the calpains’ stage and then be used as a pharmacological target. <br />
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<a name="proteolux"></a><br />
Utilization of the ProteoLux® Pro system<br />
</div><br />
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It is clearly visible that the characterization and a better comprehension of the Tau and Tau related aggregates are needed. A clear and complete understanding of those mechanisms could help to understand the disease process and could lead to new pharmacological targets. The idea is to use the ProteoLux® Pro system in order to study the mechanism of Tau fragmentation. The ProteoLux® Pro system can be implemented in cells culture from Alzheimer patients. Each protease involved in the Tau fragmentation could be studied in different cell cultures using the ProteoLux® Pro system. The protease would be fused to a Phytochrome Interacting Factor (PIF) and a degradation tag (Lambda-O in N-terminal and DAS in C-terminal). After implementation of the ClpXP system in the cell culture, the level of the targeted protease could be adjusted to a certain concentration using a succession of activating and inactivating pulses. Indeed the modeling has shown that the <b>ProteoLux® Pro system can adjust the protein concentration in the cell.</b> This allows a precise study of the targeted protease. <br />
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The utilization of the ProteoLux® Pro will without any doubt bring new insights in the mechanisms of the Alzheimer disease factors and development. It will also lead to new breakthrough in the creation of new pharmacological targets and substances. Indeed being able to control the level or even the formation of Tau aggregates might lead to cure the Alzheimer disease.<br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Neuro.pngFile:ESBS-Strasbourg-Neuro.png2010-11-30T08:27:52Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-30T02:46:19Z<p>Georgio: </p>
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
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<ul><br />
<li><a href="#degradation">Degradation System</a></li><br />
<li><a href="#light">Light detection system</a></li><br />
<li><a href="#system">Final construction</a></li><br />
<li><a href="#chassis">Introduction to various chassis</a></li><br />
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<a name="overview"></a><br />
Overview<br />
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<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
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PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
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<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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<a name="degradation"></a><br />
Degradation system<br />
</div><br />
<br><br />
<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
<br />
<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
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<br><br />
<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
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<br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
</div><br />
<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
<br><br><br />
<br />
<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
<br><br><br />
All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
<br><br><br><br><br />
<br />
<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
<br><br><br />
The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<img src="https://static.igem.org/mediawiki/2010/5/59/ESBS-Strasbourg-PhyB.png" width="192px" height="316px" align="left"><br />
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<br><br />
<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
<br><br />
We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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Final construction: Light controllable protease <br />
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PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
<br><br><br />
The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<p>&nbsp;</p><br />
<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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Introduction to various chassis <br />
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The ProteoLux® system can be applied to various chassis. To optimize the expression we developed different versions of ProteoLux® based on an improved codon usage especially adapted to the target host system : <br />
<br><br><br />
<center><br />
<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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<b>ProteoLux® bacteria</b> has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
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<b>ProteoLux® bacteria</b> has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
<br><br><br />
<br />
<b>ProteoLux® eucaria</b> has been designed for the use in eukaryotic microorganism like Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organism in molecular and cellular biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
<br><br><br />
<br />
<b>ProteoLux® eucaria</b> has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
<br><br><br />
<br />
<b>ProteoLux® mammalia<b> aims at the use in the medical research field, its codon usage has been adapted to the human species. <br />
<br><br><br />
The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
<br><br><br />
<br />
For in vivo implementations you should consider the use ProteoLux® Pro, which presents a special variant of ProteoLux® mammalia. <br />
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<br />
<br />
The introduction of ProteoLux® can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria. <br />
<br><br><br />
Mammalian species also have a ClpXP but it is expressed in mitochondria. Thus, the natural protease will not interfere with the ProteoLux® protease. Besides, it has be shown that the human ClpXP does not recognize SsrA tag and lambda Otag (Functional Proteolytic Complexes of the Human Mitochondrial ATP-dependent Protease, hClpXP (2002) J. Biol. Chem. Vol. 277, No. 23, pp. 21095–21102).<br />
<br><br><br />
<br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
<br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
<br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of ProteoLux®, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
<br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign" style="text-decoration: none"> <font color="#FF9900">targeting construct design.</font></a><br />
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<a name="scheme"></a><br />
SCHEME <br />
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To avoid this time consuming step you can resort to the ProteoLux® Pro system which further has lots of potential for in vivo studies. The details of this promising approach are explained in the following chapter. <br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overviewTeam:ESBS-Strasbourg/proteolux/overview2010-11-30T02:34:30Z<p>Georgio: </p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
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<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplusTeam:ESBS-Strasbourg/proteolux/overview/proteoluxplus2010-11-30T02:33:48Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
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<div class="desc"><br />
<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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Proteolux Plus ®<br />
</div><br />
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<br><br />
<center><br />
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<a name="Animation"></a><br />
Proteolux Plus ®<br />
</div><br />
<br><br />
<br><br />
<center><br />
<object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/pvdjXNAQMIM?hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/pvdjXNAQMIM?hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxproTeam:ESBS-Strasbourg/proteolux/overview/proteoluxpro2010-11-30T02:33:32Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
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<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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Proteolux Plus ®<br />
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<center><br />
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<a name="Animation"></a><br />
Proteolux Plus ®<br />
</div><br />
<br><br />
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<center><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/overview/proteoluxbasic2010-11-30T02:32:57Z<p>Georgio: </p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
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<br />
<br />
<li><br />
<p><br /><br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
<br />
<br />
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<div class="heading">Scientific Background</div><br />
<div class="desc"><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic">Proteolux Basic ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
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<div class="desc"><br />
<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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<a name="Animation"></a><br />
Proteolux Plus ®<br />
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Proteolux Plus ®<br />
</div><br />
<br><br />
<br><br />
<center><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/overview/proteoluxbasic2010-11-30T02:20:38Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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<a name="overview"></a><br />
Overview<br />
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<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-activeintrabody.pngFile:ESBS-Strasbourg-activeintrabody.png2010-11-30T01:49:11Z<p>Georgio: </p>
<hr />
<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-constructionab.pngFile:ESBS-Strasbourg-constructionab.png2010-11-30T01:46:51Z<p>Georgio: </p>
<hr />
<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overviewTeam:ESBS-Strasbourg/proteolux/overview2010-11-30T01:43:05Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
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<div class="heading"><br />
<a name="overview"></a><br />
Overview<br />
</div><br />
<br><br />
<br />
Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
<br><br><br />
<br />
The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
<br><br><br />
<br />
PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
<br />
<br><br><br />
<br />
Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-30T01:39:19Z<p>Georgio: </p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
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ProteOlux Plus</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
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<ul><br />
<li><a href="#degradation">Degradation System</a></li><br />
<li><a href="#light">Light detection system</a></li><br />
<li><a href="#system">Final construction</a></li><br />
<li><a href="#chassis">Introduction to various chassis</a></li><br />
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<a name="overview"></a><br />
Overview<br />
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Proteolux is a light-controllable specific protein degradation system. The system contains several parts: the bacterial ClpXP protease from E. Coli and the specific recognition sequence (DAS-tag) for ClpX for the degradation part as the photoreceptor protein phytochrome B (PhyB) and the phytochrome interacting factor 6(PIF6) from A. thaliana for the light-dependent part of the system.<br />
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The ClpXP proteases consist of three main parts: the ClpX unit and two units of ClpP. The ClpX forms a hexametric ring and binds to a double heptamer of ClpP. The ClpX is responsible for recognizing proteins bearing a specific degradation tag, unfolding and leading them into the catalytic core of the enzyme, where two ClpP subunits break down the peptides bonds. <br />
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PhyB is characterized by a red/far-red photochromicity. Through red-light absorption (650–670 nm) PhyB undergoes a rapid conformational change from its ground state Pr to its active state Pfr. The structural change allows the binding of different interacting factors (PIF).The process is completely reversible through absorption in the near infra-red spectrum (705-740nm). PhyB is fused to the N terminus of a trimeric form of ClpX that lacks the N-terminus in which the subunits were connected with a flexible linker to stabilize the enzyme. <br />
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Target proteins are fused to the PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br />
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Degradation system<br />
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<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
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<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
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<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
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Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
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<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
<br><br><br />
<br />
<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
<br><br><br />
All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
<br><br><br><br><br />
<br />
<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
<br><br><br />
The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
<br><br />
We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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<a name="system"></a><br />
Final construction: Light controllable protease <br />
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<br><br><br />
PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
<br><br><br />
The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<p>&nbsp;</p><br />
<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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<a name="chassis"></a><br />
Introduction to various chassis <br />
</div><br />
<br><br><br />
The Proteolux system can be applied to various different chassis. To optimize the expression we developed different versions of Proteolux based on an improved codon usage especially adapted to the target host system : <br />
<br><br><br />
<center><br />
<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
</ul><br />
</center><br />
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Proteolux bacteria has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
<br />
<br />
Proteolux bacteria has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
<br><br><br />
<br />
Proteolux eucaria has been designed for the use in eukaryotic microorganism as Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organisms in molecular and cell biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
<br><br><br />
<br />
Proteolux eucaria has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
<br><br><br />
<br />
Proteolux mammalia aims at the use in the medical research sector, its codon usage has been adapted to the human species. <br />
<br><br><br />
The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
<br><br><br />
<br />
For in vivo implementations you should consider the use Proteolux Pro, which presents a special variant of Proteolux mammalia. <br />
<br><br><br />
<br />
<br />
The introduction of Proteolux can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria, <br />
<br><br><br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign" style="text-decoration: none"> <font color="#FF9900">targeting construct design.</font></a><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/Team:ESBS-Strasbourg/proteolux/scientific/2010-11-30T01:28:52Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#degradation">Degradation System</a></li><br />
<li><a href="#light">Light detection system</a></li><br />
<li><a href="#system">Final construction</a></li><br />
<li><a href="#chassis">Introduction to various chassis</a></li><br />
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<a name="degradation"></a><br />
Degradation system<br />
</div><br />
<br><br />
<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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</center><br />
<br><br><br />
In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
<br />
<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2010/8/82/ESBS-Strasbourg-ImageProteinfinal%2B.png" width="239px" height="168px" align="left"><br />
<br />
<br><br />
<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
<br />
<br />
<br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
</div><br />
<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
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<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
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All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
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<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
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The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
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We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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Final construction: Light controllable protease <br />
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PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
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The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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Introduction to various chassis <br />
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The Proteolux system can be applied to various different chassis. To optimize the expression we developed different versions of Proteolux based on an improved codon usage especially adapted to the target host system : <br />
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<center><br />
<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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Proteolux bacteria has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
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Proteolux bacteria has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
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Proteolux eucaria has been designed for the use in eukaryotic microorganism as Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organisms in molecular and cell biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
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Proteolux eucaria has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
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Proteolux mammalia aims at the use in the medical research sector, its codon usage has been adapted to the human species. <br />
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The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
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For in vivo implementations you should consider the use Proteolux Pro, which presents a special variant of Proteolux mammalia. <br />
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The introduction of Proteolux can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
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In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria, <br />
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The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign" style="text-decoration: none"> <font color="#FF9900">targeting construct design.</font></a><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxproTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxpro2010-11-30T01:25:13Z<p>Georgio: </p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="desc"><br />
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<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="#proteoluxpro">Proteolux Pro ®</a></li><br />
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<a name="proteoluxp"></a><br />
Proteolux P<br />
</div><br />
<br><br />
As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system. <br />
<br><br><br />
<br />
The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.<br />
<br><br><br />
Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.<br />
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Proteolux Pro ®<br />
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<div class="desc"><br />
<br><br />
<p><b>Light-controlled regulation of the intrabody activity.</b></p><br />
<br><br />
The idea is to have an inactive intrabody which can be assembled on demand into its active form. Therefore, the structure of a general IgG antibody has to be regarded in greater detail (figure 1) [Garrett RH and Grisham CM, Biochemistry 2nd edition].<br />
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<p><span><font size="1">Figure 1: Overview over the structure of an IgG antibody. The active IgG antibody is composed of two heavy (H) chains (outer long chains) and the small light (L) chains (inner short chains). The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The Fab region which is composed of the variable domain and the CH1 domain is connected via a flexible hinge region to the Fc part of the antibody (CH2 and CH3 region). The chains are intra- and interconnected by disulfide bridges (yellow lines). There are two disulfide bridges that interconnect the two heavy chains and each variable chain is connected to one heavy chain via one disulfide bridge. The active antibody is only formed when the light chain is connected to the heavy chain in order to form the antigen binding site, which is situated between the variable domains. </font></span></p><br />
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The formation of the antigen binding site is dependent on the correct binding of the VL to the VH domain, which is enhanced by a strong affinity between the constant regions and the disulfide bridge between the light and the heavy chain. In the Proteolux Pro system, this permanent disulfide bridge is replaced by a light inducible interaction between two proteins. In order to avoid interference with the PhyB-PIF interaction, other interaction partners were searched which are active under light conditions that do not activate phytrochrome B (figure 2). <br />
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<p><span><font size="1">Figure 2: Absorption spectrum of phytrochome B containing the PCB chromophore. The absorption maxima of the two red light forms are visible as two distinct peaks at 658 nm for the red light form and at 720 nm for the far-red light form. The absorbance is lowest in the blue/green light part of the spectrum between 450nm and 550nm. Therefore, it is save to work under blue/green light conditions to avoid the activation of phytrochrome B. </font></span></p><br />
<br><br><br><br><br><br />
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The absorbance spectrum of phytrochrome B reveals that it does not absorb under light conditions between 450 nm and 550 nm which correlates to blue/green light. Therefore, the blue light (475 nm) regulated interaction between the Arabidopsis thaliana photoreceptor Zeitlupe (ZTL, Gene ID: 835842) and Gigantea (GI, Gene ID: 838883) is used to replace the disulfide bridge function. Zeitlupe contains a light sensing PAS-like LOV domain at its N-terminal end and a C-terminal Kelch repeat domain, which is involved in protein-protein interaction [Lariguet P and Dunand C, 2005; http://www.uniprot.org/uniprot/Q94BT6]. The GI protein has a more than 4 fold affinity to ZTL under blue light conditions [Kim WY, 2007].<br><br />
In order to prevent the strong affinity between the constant domains of the light and the heavy chain, only the low affinity variable domains are used in the construction. <br><br />
The ZTL sequence is fused together with the normal PIF-DAS-tag to the C-terminus of the VL domain and the GI sequence followed by a GST sequence to the C-terminus of the VH domain. The GST dimerizes and connects therefore two VH domains. This allows the formation of bivalent intrabodies with higher protein binding capacity.<br><br />
The expression of such a modified intrabody allows the formation of the active intrabody only under blue light conditions. The protein expressing phenotype can be analyzed under far-red light conditions. When the protein should be degraded, the intrabody formation is triggered under blue light (475nm) resulting in the formation of an active antibody binding site and fixation of the target protein by the intrabody. To ensure, that the protein is not active anymore the intrabody-protein complex can be tethered to the protease using red light conditions. This procedure is summarized in figure 3. <br><br><br />
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<center><br />
<p><b>Proteolux Pro offers the first light-controlled intrabody activity which can be used to analyze any protein in its native conformation without interference with the cell metabolism. </b></p></center><br />
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<font size="1">Figure 3: Light controlled intrabody formation and specific target protein degradation.<br />
A: Structure of an IgG antibody. Shown are the two long heavy (H) chains and the small light (L) chains. The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The chains are intra- and interconnected by disulfide bridges (yellow lines). B1.1 and B1.2: Fusion constructs for the light and heavy chains. B1.1: The blue light photoreceptor Zeitlupe (ZTL), the Phytochrome interacting factor (PIF) and the C-terminal degradation tag DAS are fused to the C-terminus of the light chain. B1.2: The gigantea (GI) sequence is fused in the hinge region of the antibody. The two conserved cysteine residues for the disulfide bridge are maintained, the two constant domains (CH2 and CH3) of the heavy chain are deleted. C: Blue light dependent photo conversion of ZTL. The absorption of blue light (475 nm) by the PAS-like LOV domain of ZTL leads to a conformational change allowing GI binding (D). D: Blue light induced formation of the active intrabody. The binding of GI to ZTL under blue light brings the light and heavy chain together and restores the natural disulfide bridge between the two. The two variable domains can now form an active antigen binding site that binds to the protein of interest. E: Red light induced protein degradation. PIF binds to PhyB under red light conditions bringing the DAS tag in proximity to the ClpXP protease leading to degradation of the protein of interest bound to the intrabody.</font></div><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxproTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxpro2010-11-30T01:22:45Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
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<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="#proteoluxpro">Proteolux Pro ®</a></li><br />
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Proteolux P<br />
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As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system. <br />
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The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.<br />
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Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.<br />
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Proteolux Pro ®<br />
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<div class="desc"><br />
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<p><b>Light-controlled regulation of the intrabody activity.</b></p><br />
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The idea is to have an inactive intrabody which can be assembled on demand into its active form. Therefore, the structure of a general IgG antibody has to be regarded in greater detail (figure 1) [Garrett RH and Grisham CM, Biochemistry 2nd edition].<br />
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<p><span><font size="1">Figure 1: Overview over the structure of an IgG antibody. The active IgG antibody is composed of two heavy (H) chains (outer long chains) and the small light (L) chains (inner short chains). The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The Fab region which is composed of the variable domain and the CH1 domain is connected via a flexible hinge region to the Fc part of the antibody (CH2 and CH3 region). The chains are intra- and interconnected by disulfide bridges (yellow lines). There are two disulfide bridges that interconnect the two heavy chains and each variable chain is connected to one heavy chain via one disulfide bridge. The active antibody is only formed when the light chain is connected to the heavy chain in order to form the antigen binding site, which is situated between the variable domains. </font></span></p><br />
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The formation of the antigen binding site is dependent on the correct binding of the VL to the VH domain, which is enhanced by a strong affinity between the constant regions and the disulfide bridge between the light and the heavy chain. In the Proteolux Pro system, this permanent disulfide bridge is replaced by a light inducible interaction between two proteins. In order to avoid interference with the PhyB-PIF interaction, other interaction partners were searched which are active under light conditions that do not activate phytrochrome B (figure 2). <br />
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<p><span><font size="1">Figure 2: Absorption spectrum of phytrochome B containing the PCB chromophore. The absorption maxima of the two red light forms are visible as two distinct peaks at 658 nm for the red light form and at 720 nm for the far-red light form. The absorbance is lowest in the blue/green light part of the spectrum between 450nm and 550nm. Therefore, it is save to work under blue/green light conditions to avoid the activation of phytrochrome B. </font></span></p><br />
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The absorbance spectrum of phytrochrome B reveals that it does not absorb under light conditions between 450 nm and 550 nm which correlates to blue/green light. Therefore, the blue light (475 nm) regulated interaction between the Arabidopsis thaliana photoreceptor Zeitlupe (ZTL, Gene ID: 835842) and Gigantea (GI, Gene ID: 838883) is used to replace the disulfide bridge function. Zeitlupe contains a light sensing PAS-like LOV domain at its N-terminal end and a C-terminal Kelch repeat domain, which is involved in protein-protein interaction [Lariguet P and Dunand C, 2005; http://www.uniprot.org/uniprot/Q94BT6]. The GI protein has a more than 4 fold affinity to ZTL under blue light conditions [Kim WY, 2007].<br><br />
In order to prevent the strong affinity between the constant domains of the light and the heavy chain, only the low affinity variable domains are used in the construction. <br><br />
The ZTL sequence is fused together with the normal PIF-DAS-tag to the C-terminus of the VL domain and the GI sequence followed by a GST sequence to the C-terminus of the VH domain. The GST dimerizes and connects therefore two VH domains. This allows the formation of bivalent intrabodies with higher protein binding capacity.<br><br />
The expression of such a modified intrabody allows the formation of the active intrabody only under blue light conditions. The protein expressing phenotype can be analyzed under far-red light conditions. When the protein should be degraded, the intrabody formation is triggered under blue light (475nm) resulting in the formation of an active antibody binding site and fixation of the target protein by the intrabody. To ensure, that the protein is not active anymore the intrabody-protein complex can be tethered to the protease using red light conditions. This procedure is summarized in figure 3. <br><br><br />
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<p><b>Proteolux Pro offers the first light-controlled intrabody activity which can be used to analyze any protein in its native conformation without interference with the cell metabolism. </b></p></center><br />
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<font size="1">Figure 3: Light controlled intrabody formation and specific target protein degradation.<br />
A: Structure of an IgG antibody. Shown are the two long heavy (H) chains and the small light (L) chains. The constant domains are shown in green (C) and the variable domains in violet (V). The heavy chains consist of 3 constant domains (CH1, CH2 and CH3) and a variable domain VH. The light chains are composed of a constant (CL) and a variable (VL) domain. The chains are intra- and interconnected by disulfide bridges (yellow lines). B1.1 and B1.2: Fusion constructs for the light and heavy chains. B1.1: The blue light photoreceptor Zeitlupe (ZTL), the Phytochrome interacting factor (PIF) and the C-terminal degradation tag DAS are fused to the C-terminus of the light chain. B1.2: The gigantea (GI) sequence is fused in the hinge region of the antibody. The two conserved cysteine residues for the disulfide bridge are maintained, the two constant domains (CH2 and CH3) of the heavy chain are deleted. C: Blue light dependent photo conversion of ZTL. The absorption of blue light (475 nm) by the PAS-like LOV domain of ZTL leads to a conformational change allowing GI binding (D). D: Blue light induced formation of the active intrabody. The binding of GI to ZTL under blue light brings the light and heavy chain together and restores the natural disulfide bridge between the two. The two variable domains can now form an active antigen binding site that binds to the protein of interest. E: Red light induced protein degradation. PIF binds to PhyB under red light conditions bringing the DAS tag in proximity to the ClpXP protease leading to degradation of the protein of interest bound to the intrabody.</font></div><br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-proteoluxpro.pngFile:ESBS-Strasbourg-proteoluxpro.png2010-11-30T01:17:12Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-absorption.pngFile:ESBS-Strasbourg-absorption.png2010-11-30T01:01:07Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-intracorps.pngFile:ESBS-Strasbourg-intracorps.png2010-11-30T00:58:56Z<p>Georgio: uploaded a new version of "Image:ESBS-Strasbourg-intracorps.png"</p>
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<div></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-intracorps.pngFile:ESBS-Strasbourg-intracorps.png2010-11-30T00:46:54Z<p>Georgio: </p>
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<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplusTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxplus2010-11-30T00:41:59Z<p>Georgio: </p>
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<br><br />
<div class="heading">ProteOlux ®</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="#proteoluxplus">Proteolux Plus ®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro ®</a></li><br />
<br />
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<a name="proteoluxp"></a><br />
Proteolux P<br />
</div><br />
<br><br />
As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system. <br />
<br><br><br />
<br />
The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.<br />
<br><br><br />
Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.<br />
<br />
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<div class="heading"><br />
<a name="proteoluxplus"></a><br />
Proteolux Plus ®<br />
</div><br />
<div class="desc"><br />
<br><br />
<p><b>Intrabody expression regulation by Proteolux’s light inducible degradation system.</b></p><br />
<br><br />
It has been shown (A-P.Sibler & al. 2005) that the C-terminus fusion of a PEST sequence (a protein region rich in proline, glutamic acid, serine and threonine) to an intrabody is a key element for binding to the control region of the 26S proteasomes (Zhang M. & al. 2003). This modification leads to a shortened half-life of the intrabody which is transferred to the bound target protein as it is degraded together with the intrabody. These PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools to shorten/decrease the half-life of the target protein.<br />
<br><br><br />
For the controlled regulation of the target protein, the destabilized intrabody is controlled by a strong eukaryotic promoter, which is repressed by a strong repressor. The used repressor is tagged with the PIF-DAS tag construct and is constitutively expressed. Consequently the expression of the destabilized intrabody will be repressed until the repressor is degraded by a red light pulse. Once the intrabody containing the PEST sequence is expressed, it binds to the target protein and targets the bound protein to the proteasome. The intrabody is expressed continuously until the degradation of the repressor degradation is stopped by a far-red light pulse. <br />
<br><br><br />
<br />
<center><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplusTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxplus2010-11-30T00:41:16Z<p>Georgio: </p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
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<div class="desc"><br />
<br />
<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="#proteoluxplus">Proteolux Plus®</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro®</a></li><br />
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<div class="heading"><br />
<a name="proteoluxp"></a><br />
Proteolux P<br />
</div><br />
<br><br />
As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system. <br />
<br><br><br />
<br />
The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.<br />
<br><br><br />
Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.<br />
<br />
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<a name="proteoluxplus"></a><br />
Proteolux Plus ®<br />
</div><br />
<div class="desc"><br />
<br><br />
<p><b>Intrabody expression regulation by Proteolux’s light inducible degradation system.</b></p><br />
<br><br />
It has been shown (A-P.Sibler & al. 2005) that the C-terminus fusion of a PEST sequence (a protein region rich in proline, glutamic acid, serine and threonine) to an intrabody is a key element for binding to the control region of the 26S proteasomes (Zhang M. & al. 2003). This modification leads to a shortened half-life of the intrabody which is transferred to the bound target protein as it is degraded together with the intrabody. These PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools to shorten/decrease the half-life of the target protein.<br />
<br><br><br />
For the controlled regulation of the target protein, the destabilized intrabody is controlled by a strong eukaryotic promoter, which is repressed by a strong repressor. The used repressor is tagged with the PIF-DAS tag construct and is constitutively expressed. Consequently the expression of the destabilized intrabody will be repressed until the repressor is degraded by a red light pulse. Once the intrabody containing the PEST sequence is expressed, it binds to the target protein and targets the bound protein to the proteasome. The intrabody is expressed continuously until the degradation of the repressor degradation is stopped by a far-red light pulse. <br />
<br><br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b6/ESBS-Strasbourg-Proteoluxplus.png" width="400px" height="235px" align="center"> </center><br />
<center> The functioning of the Proteolux Plus </center> <br />
<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplusTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxplus2010-11-30T00:37:29Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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ProteOlux Pro</a></li><br />
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">ProteOlux</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#proteoluxp">Proteolux P</a></li><br />
<li><a href="#proteoluxplus">Proteolux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro#proteoluxpro">Proteolux Pro</a></li><br />
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<a name="proteoluxp"></a><br />
Proteolux P<br />
</div><br />
<br><br />
As shown previously the Proteolux system offers a light-inducible specific mechanism to degrade tagged protein. This system may be applied to any protein expressed in the cytoplasm of the host cell. For the original system a homologue recombination step is necessary in order to fuse the PIF-DAS tag to the target protein in the host cell or organism. This step requires additional time in order to prepare the experiment and might further change the normal protein physiology, which should be observed, due to the creation of a fusion protein. It is known, that the function of a fusion protein can be different of the wild-type protein due to different stability of the mRNA after the homologue recombination leading to a different translation rate or the expressed protein does not fold correctly and forms aggregates in the cytoplasm. These drawbacks of the original system are solved in Proteolux’s P (Plus and Pro) system. <br />
<br><br><br />
<br />
The Proteolux Plus and Pro system allows the observation of the native target protein functions without long experiment preparation and interference with the cell metabolism. The developed strategy is to use a specifically engineered intrabody against the target protein. The binding of the intrabody to the protein can lead to the direct inhibition of the protein function. In the case, that the protein is not directly inhibited and to prevent re-establishment of the protein function the intrabody-protein complex is targeted to the proteasome. The binding of the intrabody to the target protein has to be regulated to avoid constant degradation of the target protein. Therefore, Proteolux offers the Plus and Pro system of different complexity, according to the experimenter demand.<br />
<br><br><br />
Intrabodies are antibody derived proteins that bind to an intracellular protein within the cell. The normal antibody (figure X) has to be modified due to the reducing environment within the cytoplasm that prevents disulfide bridge formation. Therefore, single-chain antibodies, which are fusion proteins of the variable region of the light chain (VL) and the heavy chain (VH) connected by a short linker, are often expressed.<br />
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<a name="proteoluxplus"></a><br />
Proteolux Plus<br />
</div><br />
<div class="desc"><br />
<br><br />
<p><b>Intrabody expression regulation by Proteolux’s light inducible degradation system.</b></p><br />
<br><br />
It has been shown (A-P.Sibler & al. 2005) that the C-terminus fusion of a PEST sequence (a protein region rich in proline, glutamic acid, serine and threonine) to an intrabody is a key element for binding to the control region of the 26S proteasomes (Zhang M. & al. 2003). This modification leads to a shortened half-life of the intrabody which is transferred to the bound target protein as it is degraded together with the intrabody. These PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools to shorten/decrease the half-life of the target protein.<br />
<br><br><br />
For the controlled regulation of the target protein, the destabilized intrabody is controlled by a strong eukaryotic promoter, which is repressed by a strong repressor. The used repressor is tagged with the PIF-DAS tag construct and is constitutively expressed. Consequently the expression of the destabilized intrabody will be repressed until the repressor is degraded by a red light pulse. Once the intrabody containing the PEST sequence is expressed, it binds to the target protein and targets the bound protein to the proteasome. The intrabody is expressed continuously until the degradation of the repressor degradation is stopped by a far-red light pulse. <br />
<br><br><br />
<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2010/b/b6/ESBS-Strasbourg-Proteoluxplus.png" width="400px" height="235px" align="center"> </center><br />
<center> The functioning of the Proteolux Plus </center> <br />
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</html></div>Georgiohttp://2010.igem.org/File:ESBS-Strasbourg-Proteoluxplus.pngFile:ESBS-Strasbourg-Proteoluxplus.png2010-11-30T00:30:31Z<p>Georgio: </p>
<hr />
<div></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesignTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign2010-11-30T00:11:57Z<p>Georgio: </p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
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<div class="desc"><br />
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<ul><br />
<li><a href="#targeting">Gene Targeting Constructs</a></li><br />
<li><a href="#homology">Homology Arms</a></li><br />
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<a name="targeting"></a><br />
Gene Targeting Constructs<br />
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<br><br />
Gene targeting contructs are designed to undergo homologous recombination into a specific locus chosen by the investigator, usually with the aim of disrupting the gene to prevent transcription of a functional mRNA (a knock-out), or mutating the gene (a knock-in).<br />
<br><br><br />
The number of ways in which gene targeting constructs can be designed to produce knock-out or knock-in is almost limitless. Thus, we can only offer some general guidelines here, and urge people making their first construct to read the literature and consult with the TMF or another more experienced investigator before doing any cloning.<br />
<br><br><br />
The simplest targeting construct consists of 2 long segments of genomic DNA (gDNA), called homology arms, flanking a selection cassette. The most commonly used selection cassette consists of the cDNA and control elements for the neomycin (G418) resistance gene (others include resistance genes for puromycin, hygromycin, and 6-thioguanine).<br />
<br><br><br />
When introduced into the cell line, the gDNA homology arms will undergo recombination with their matching sequences on one chromosome, carrying the selection cassette with them. The gDNA between the regions of homology on the chromosome is thereby replaced by the selection cassette and any other sequences flanked by the homology arms of the targeting construct. Where a complete knockout is desired, the intervening sequence is usually positioned to replace the TATA box, the start codon, and one or more of the initial exons.<br />
<br><br><br />
The targeting construct will also integrate into random loci. Any integration event, random or specific, can confer drug resistance to the cell. After growing the transfected cells under selection, the challenge is to screen enough clones to find the rare homologous recombination events in a background of frequent random integrants.<br />
<br><br><br />
<b>We recommend the use of both positive and negative selection cassettes in all targeting constructs.</b> The most commonly used negative selection cassette contains the gene for thymidine kinase, or tk. The tk gene product allows growing cells to incorporate a toxic nucleotide analog into their DNA, thus selecting against those cells. The tk cassette is cloned into the targeting construct outside of the homology arms, so that it <b>will</b> not be incorporated during homologous recombination. It will be incorporated during random integration and help to select against those clones. Another negatively selectable marker is the gene for diphtheria toxin A. The A subunit inhibits protein synthesis but cannot be taken up by other cells. Its advantage over tk is that it works without having to add a second drug to the culture medium.<br />
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<a name="homology"></a><br />
Homology Arms<br />
</div><br />
<div class="desc"><br />
<br><br />
The degree to which the homology arms match the same sequences in the locus of interest will help determine the frequency of homologous recombination. The 3 most important characteristics of homology arms are:<br />
<br><br />
<br><br />
<br />
<ol><br />
<li>Length – we recommend an overall length of about 7 kilobases, with one arm being 5-6 kb and the other being 1-2 kb. Longer is better, but one is usually limited by the capacity of the cloning vector and the need to maintain a unique restriction enzyme site that can be used to linearize the construct prior to transfection into the cells.</li><br />
<br><br />
<li>Sequence homology – whenever possible, clone the homology arms from the genome of the cells that will be targeted. Long-range PCR with a high-fidelity polymerase is an effective method for subcloning the homology arms.</li><br />
<br><br />
<li>Limited repetitive sequences – we recommend using the on-line program, RepeatMasker, to search for repetitive sequences in the homology arms. Large regions of repetitive DNA should be avoided, because these will result in a lower frequency of homologous recombination.</li><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesignTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign2010-11-30T00:02:20Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br /><br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
<br />
<br />
</ul><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#targeting">Gene Targeting Constructs</a></li><br />
<li><a href="#homology">Homology Arms</a></li><br />
<br />
<br />
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<br />
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</p></div><br />
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<div class="heading"><br />
<a name="targeting"></a><br />
Gene Targeting Constructs<br />
</div><br />
<br><br />
Gene targeting contructs are designed to undergo homologous recombination into a specific locus chosen by the investigator, usually with the aim of disrupting the gene to prevent transcription of a functional mRNA (a knock-out), or mutating the gene (a knock-in).<br />
<br><br><br />
The number of ways in which gene targeting constructs can be designed to produce knock-out or knock-in is almost limitless. Thus, we can only offer some general guidelines here, and urge people making their first construct to read the literature and consult with the TMF or another more experienced investigator before doing any cloning.<br />
<br><br><br />
The simplest targeting construct consists of 2 long segments of genomic DNA (gDNA), called homology arms, flanking a selection cassette. The most commonly used selection cassette consists of the cDNA and control elements for the neomycin (G418) resistance gene (others include resistance genes for puromycin, hygromycin, and 6-thioguanine).<br />
<br><br><br />
When introduced into the cell line, the gDNA homology arms will undergo recombination with their matching sequences on one chromosome, carrying the selection cassette with them. The gDNA between the regions of homology on the chromosome is thereby replaced by the selection cassette and any other sequences flanked by the homology arms of the targeting construct. Where a complete knockout is desired, the intervening sequence is usually positioned to replace the TATA box, the start codon, and one or more of the initial exons.<br />
<br><br><br />
The targeting construct will also integrate into random loci. Any integration event, random or specific, can confer drug resistance to the cell. After growing the transfected cells under selection, the challenge is to screen enough clones to find the rare homologous recombination events in a background of frequent random integrants.<br />
<br><br><br />
<b>We recommend the use of both positive and negative selection cassettes in all targeting constructs.</b> The most commonly used negative selection cassette contains the gene for thymidine kinase, or tk. The tk gene product allows growing cells to incorporate a toxic nucleotide analog into their DNA, thus selecting against those cells. The tk cassette is cloned into the targeting construct outside of the homology arms, so that it <b>will</b> not be incorporated during homologous recombination. It will be incorporated during random integration and help to select against those clones. Another negatively selectable marker is the gene for diphtheria toxin A. The A subunit inhibits protein synthesis but cannot be taken up by other cells. Its advantage over tk is that it works without having to add a second drug to the culture medium.<br />
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<div class="heading"><br />
<a name="homology"></a><br />
Homology Arms<br />
</div><br />
<div class="desc"><br />
<br><br />
The degree to which the homology arms match the same sequences in the locus of interest will help determine the frequency of homologous recombination. The 3 most important characteristics of homology arms are:<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-29T23:55:16Z<p>Georgio: </p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#degradation">Degradation System</a></li><br />
<li><a href="#light">Light detection system</a></li><br />
<li><a href="#system">Final construction</a></li><br />
<li><a href="#chassis">Introduction to various chassis</a></li><br />
<br />
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<div class="heading"><br />
<a name="degradation"></a><br />
Degradation system<br />
</div><br />
<br><br />
<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
<br><br><br />
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<br><br><br />
In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
<br />
<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
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<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
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Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
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<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
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<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
<br><br><br />
All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
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<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
<br><br><br />
The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
<br><br />
We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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<a name="system"></a><br />
Final construction: Light controllable protease <br />
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PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
<br><br><br />
The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<p>&nbsp;</p><br />
<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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<a name="chassis"></a><br />
Introduction to various chassis <br />
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The Proteolux system can be applied to various different chassis. To optimize the expression we developed different versions of Proteolux based on an improved codon usage especially adapted to the target host system : <br />
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<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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Proteolux bacteria has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
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Proteolux bacteria has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
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Proteolux eucaria has been designed for the use in eukaryotic microorganism as Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organisms in molecular and cell biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
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Proteolux eucaria has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
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Proteolux mammalia aims at the use in the medical research sector, its codon usage has been adapted to the human species. <br />
<br><br><br />
The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
<br><br><br />
<br />
For in vivo implementations you should consider the use Proteolux Pro, which presents a special variant of Proteolux mammalia. <br />
<br><br><br />
<br />
<br />
The introduction of Proteolux can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria, <br />
<br><br><br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign" style="text-decoration: none"> <font color="#FF9900">targeting construct design.</font></a><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-29T23:50:45Z<p>Georgio: </p>
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<br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br /><br />
<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
<br />
<br />
</ul><br />
</li><br />
<br />
<br />
<li><br />
<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#Animation">Animation</a></li><br />
<li><a href="#molstruc">Molecular Structure of the System</a></li><br />
<li><a href="#molstrucunit">Molecular Structure of 1 unit</a></li><br />
<li><a href="#system">System description</a></li><br />
<li><a href="#assembly">Assembly</a></li><br />
<li><a href="#taggedprot">Tagged protein</a></li><br />
<li><a href="#mecadesc">Mechanical description</a></li><br />
<li><a href="#workstation">Workstation</a></li><br />
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<a name="degradation"></a><br />
Degradation system<br />
</div><br />
<br><br />
<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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<br><br><br />
In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
<br />
<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
<br><br><br />
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<br />
<br><br />
<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
<br />
<br />
<br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
</div><br />
<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
<br><br><br />
<br />
<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
<br><br><br />
All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<img src="https://static.igem.org/mediawiki/2010/c/c3/ESBS-Strasbourg-Phybdomain.png" width="148px" height="270px" align="left"><br />
<br><br />
<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
<br><br><br><br><br />
<br />
<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
<br><br><br />
The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
<br><br><br />
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<img src="https://static.igem.org/mediawiki/2010/5/59/ESBS-Strasbourg-PhyB.png" width="192px" height="316px" align="left"><br />
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<br><br />
<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
<br><br />
We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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<div class="heading"><br />
<a name="system"></a><br />
Final construction: Light controllable protease <br />
</div><br />
<br><br><br />
PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
<br><br><br />
The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<img src="https://static.igem.org/mediawiki/2010/8/87/ESBS-Strasbourg-System.png" width="192px" height="317px" align="left"><br />
<p>&nbsp;</p><br />
<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
</span></p><br />
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<a name="chassis"></a><br />
Introduction to various chassis <br />
</div><br />
<br><br><br />
The Proteolux system can be applied to various different chassis. To optimize the expression we developed different versions of Proteolux based on an improved codon usage especially adapted to the target host system : <br />
<br><br><br />
<center><br />
<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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</center><br />
<br><br><br />
<br />
<br />
Proteolux bacteria has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
<br />
<br />
Proteolux bacteria has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
<br><br><br />
<br />
Proteolux eucaria has been designed for the use in eukaryotic microorganism as Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organisms in molecular and cell biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
<br><br><br />
<br />
Proteolux eucaria has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
<br><br><br />
<br />
Proteolux mammalia aims at the use in the medical research sector, its codon usage has been adapted to the human species. <br />
<br><br><br />
The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
<br><br><br />
<br />
For in vivo implementations you should consider the use Proteolux Pro, which presents a special variant of Proteolux mammalia. <br />
<br><br><br />
<br />
<br />
The introduction of Proteolux can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria, <br />
<br><br><br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on <a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic/constructdesign"><br />
targeting construct design.</a><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-29T23:47:41Z<p>Georgio: </p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#Animation">Animation</a></li><br />
<li><a href="#molstruc">Molecular Structure of the System</a></li><br />
<li><a href="#molstrucunit">Molecular Structure of 1 unit</a></li><br />
<li><a href="#system">System description</a></li><br />
<li><a href="#assembly">Assembly</a></li><br />
<li><a href="#taggedprot">Tagged protein</a></li><br />
<li><a href="#mecadesc">Mechanical description</a></li><br />
<li><a href="#workstation">Workstation</a></li><br />
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<a name="degradation"></a><br />
Degradation system<br />
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<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
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<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
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<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br><br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
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<br />
<br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. The C-terminal fusion of PIF6 using the N-terminal degradation tag λO is consequently more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO –tag) which do not perturb protein activity in most of the cases.<br><br><br />
Nevertheless, if possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux.<br />
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<a name="light"></a><br />
Light detection system<br />
</div><br />
<div class="desc"><br />
<br><br />
As previously mentioned, the Phytochrome/PIF system has been chosen as light detection system. There are several advantages of this system: it offers a second timescale control which is significantly faster than chemically induced translocation systems, further it is perfectly reversible as it has been proven to be robust being cycled over a hundred times by alternating red and infrared illumination with no measurable decrease in recruitment ratios over time [41]. <br />
<br><br><br />
<br />
<p><b>Description</b></p><br />
Phytochromes are photoreceptive signaling proteins responsible for mediating many light-sensitive processes in plants. They detect red and near-infrared light through the photoisomerization of a covalently bound tetrapyrrole chromophore such as phycocyanobilin (PCB) for plant phytochromes. This photoisomerization event is coupled to an allosteric transition in the phytochrome between two conformational states called Pr (red-absorbing) and Pfr (far-red-absorbing). <br><br><br />
Upon stimulation with red light (650 nm), the phytochrome B (PhyB) protein binds directly to a downstream transcription factor, the phytochrome interaction factor (PIF). PIF is a nuclear-localized, basic helix–loop–helix (bHLH) factor initially isolated as interacting with the non-photoactive, C-terminal domain of Arabidopsis PhyB. The process is completely reversible through absorption in the near infra-red spectrum (705-740nm).<br />
<br><br><br />
All plant phytochromes can be divided into an N-terminal photo sensory domain and a C-terminal dimerization domain. The N-terminal photo sensory domain comprises four consecutive subdomains called P1, P2/PAS, P3/GAF, and P4/PHY (named sequentially from the N terminus), the C-terminal domain consists of two subdomains, the PAS-A and PAS-B domains and the histidine kinase–related domain (HKRD) [45].<br />
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<p><span>The P1 domain is not essential for the function of PHYB. Deletion of amino acids 1–57 of Arabidopsis PHYB yields a protein with full activity [30]. In contrast, the P2/PAS and P3/GAF domains form the essential photo sensory core domain. These domains contain a bilin lyase activity, which is responsible for the binding of the chromophore to a cysteine residue in the P3/GAF domain. The P2/PAS and P3/GAF domains play critical roles in photo sensing, whereas the P4/PHY domain is necessary for fine tuning phytochrome activity. The autophosphorylation and phytochrome-directed phosphorylation of other proteins, such as PIF3 is attributed to a serine/threonine kinase domain located in the N-terminal [1]. Deletion of the P4/PHY domain increases the dark reversion rate (i.e., the instability of the Pfr conformation) and causes a blue shift in absorption by both Pr and Pfr. The PAS-A and PAS-B domains of PHYB are necessary for dimerization and nuclear localization, whereas PAS-A, PAS-B, and the HKRD domains are necessary for nuclear speckle formation [1]. Dimerization is required for PhyB full activity. </span></p><br />
<br><br><br><br><br />
<br />
<p><b>Construction Choice</b></p><br />
To avoid potential sterical hindrance for ClpX activity we attempted to reduce its size as much as possible.<br />
<br><br><br />
The light-sensitive interaction between PHYB and PIF3 has been mapped to the 650-residue amino- terminal photosensory core of PHYB [30]. The improved understanding of these mechanisms has been helpful for the design of the first engineered photoreceptors that utilized the interaction between PhyB and PIF3 to achieve light regulation of target proteins.<br />
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<img src="https://static.igem.org/mediawiki/2010/5/59/ESBS-Strasbourg-PhyB.png" width="192px" height="316px" align="left"><br />
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<p><span>Leung et al. put the association of the GTPase Cdc42 with its effector protein WASP under red-light control. When in complex with PhyB-Cdc42, PIF3-WASP promoted actin polymerization in vitro; use in vivo was not demonstrated. Lastly, Voigt and colleges employed the light-dependent interaction between PhyB and PIF6 to activate target proteins in vivo. Thereby they have shown that the PIF-interaction with the PhyB photo sensory core (residues 1–642) is irreversible in infrared light. By assaying PIF6, which has the strongest interactions of all previously reported PIF domains, against different variants of PhyB they demonstrated that the tandem C-terminal PAS domains (residues 1-908) of PhyB are necessary to confer rapid photo reversibility under infrared light. </span></p><br />
<br><br />
We have tested different variants for the implementation of our system: the residues 1-908 and the photo sensory core domain residues 1-642 for the phytochrome B constructs in combination with PIF3 (residues 1-100) and PIF6 (residues 1-100).<br><br><br><br><br><br />
Our tests confirmed the findings of Voigt et al. that the tandem C-terminal PAS domains of PhyB are necessary to maintain the reversibility of the system. The Proteolux system therefore uses the PhyB residues 1-908 in combination with PIF6. The restriction of enzyme activity due to sterical hindrances could be reduced to less than 5% for the optimized final system. <br><br><br />
<br />
<p><b>The chromophore</b></p><br />
<br />
Since the plant phytochromes PhyA and PhyB employ the modified tetrapyrroles PCB or PΦB which are not available in most tissues and cell types, these chromophores must be supplied either exogenously or endogenously. It is possible to produce the holophytochrome in E. coli by co-expressing two genes from Synechocystis for chromophore biosynthesis together withcyanobacterial chromophore 1(Cph1) from the same organism [15],[35]. Heme oxygenase converts host heme to biliverdin IXK which is then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase. The Cph1 apophytochrome is able to autoassemble with the phycocyanobilin in vivo to form the fully photoreversible holophytochrome.<br><br><br />
Nevertheless, we decided to use the exogenous way by adding exogenous PCB as the endogenous way to produce the holoenzyme can lead to the production of toxic side-products. <br />
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<a name="system"></a><br />
Final construction: Light controllable protease <br />
</div><br />
<br><br><br />
PhyB is fused to the N terminus of a trimeric form of ClpX-N in which the subunits were connected with a flexible linker to stabilize the enzyme. So the ClpX-hexamer is composed of two trimers fused to PhyB at its N-terminal.<br />
<br><br><br />
The reason for this particular design is that N-domain dimerization is needed to stabilize the active hexameric form of ClpX [14]. So replacing this domain, as in our construction, would result in weaker hexamerization. <br><br><br />
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<p>&nbsp;</p><br />
<br><br><br />
<p><span>In our system the adaptor-role is assumed by PIF, which will bind to PhyB following light-induction. Target proteins are fused to PIF and tagged with the specific degradation sequence which, through light activation, brings the degradation sequence in proximity to ClpX and guides them to the catalytic core of the protease. Therefore a specific degradation of proteins containing the degradation sequence can be induced by a light signal.<br><br> <br />
The system can be constitutively expressed in the chassis but it remains inactive until light-induction. However, it is expected to stay active for the background of naturally SsrA-tagged proteins, creating no interference with the natural occurring proteins.<br />
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<a name="chassis"></a><br />
Introduction to various chassis <br />
</div><br />
<br><br><br />
The Proteolux system can be applied to various different chassis. To optimize the expression we developed different versions of Proteolux based on an improved codon usage especially adapted to the target host system : <br />
<br><br><br />
<center><br />
<ul><br />
<li>Proteolux bacteria </li><br />
<li>Proteolux eucaria </li><br />
<li>Proteolux mammalia </li><br />
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<br />
<br />
Proteolux bacteria has been designed for an optimized expression in bacteria, the codon usage is primarily adapted to the class of proteobacteria. Proteobacteria are a major group of bacteria. They include a wide variety of pathogens, such as Escherichia, Salmonella, Vibrio, Helicobacter and many other notable genera. Others are free-living, and include many of the bacteria responsible for nitrogen fixation or involved in the carbon cycle. <br><br><br />
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Proteolux bacteria has been successfully implemented in various representatives of proteobacteria, including Caulobacter crescentus, Escherichia Coli, Pseudomonas fluorescens , Salmonella enterica and Vibrio fischeri.<br />
<br><br><br />
<br />
Proteolux eucaria has been designed for the use in eukaryotic microorganism as Saccharomyces cerevisiae that represents one of the most intensively studied unicellular eukaryotic model organisms in molecular and cell biology. Many proteins important in human biology were first discovered by studying their homologs in S. cerevisiae; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes. <br />
<br><br><br />
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Proteolux eucaria has further been tested in Schizosaccharomyces pombe, another yeast species and Ashbya gossypi, a model organism for filamentous fungi. <br />
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<br />
Proteolux mammalia aims at the use in the medical research sector, its codon usage has been adapted to the human species. <br />
<br><br><br />
The system can be implemented in different mammalian cell lines in vitro, including the human cell lines HEK293, K562 and HeLa, embryonic mouse fibroblast cells 3T3 as the baby hamster kidney cell line BHK-21. <br />
<br><br><br />
<br />
For in vivo implementations you should consider the use Proteolux Pro, which presents a special variant of Proteolux mammalia. <br />
<br><br><br />
<br />
<br />
The introduction of Proteolux can be realized with common transformation, transfection or transduction methods. It requires the introduction of the light inducible degradation complex ClpX-PhyB as the ClpP-gene for non-bacterial systems. <br><br><br />
<br />
In proteobacteria species encode the ClpXP protease in their proper genome. The naturally present ClpP-subunit assembles auto-catalytically with ClpX, therefore the ClpP-gene has not to be added in proteobacteria, <br />
<br><br><br />
The target gene needs to be fused with Pif6 and the specific degradation sequence. We provide two different variants to avoid problems with the accessibility or an increased activity of the target protein: The N-terminally fused λO-tag, resulting in the target protein construct [λO-protein-linker-PIF6], and the C-terminal degradation tag DAS, resulting in the target protein construct [PIF6-linker-Protein-DAS]. <br />
<br><br><br />
Most bacterial proteins tolerate C-terminal fusions and the C-termini of most proteins are solvent accessible. Consequently, the C-terminal fusion of Pif6 using the N-terminal degradation tag λO is more promising to succeed, as the specific degradation sequence consists of very few amino acids (3 for the DAS-tag and 11 for the λO–tag) which do not perturb protein activity in most of the cases. <br><br><br />
If possible, we recommend to test the activity of the target protein within the two constructs before the implementation of Proteolux, as the integration of the Pif6- and degradation tag-flanked target protein cassette presents a more time-consuming step for analytical purposes. <br><br><br />
The analysis of a specific target protein requires its fusion to the adaptor Pif6 and the specific degradation tag λO/DAS. To simplify the addition of these two sequences to your target gene we provide a special plasmid containing standard restriction sides for the integration of the target gene. Thus the integration requires no more that the amplification of your target gene with the standard restriction sites and its ligation into the plasmid. <br />
<br><br><br />
To avoid interferences with the original protein it is necessary to silence the native gene. Therefore it is necessary to a gene-knock-out using the common gene targeting approaches based on homologous recombination. We recommend to integrate the Pif6- and degradation tag-flanked target protein cassette directly into the native gene or to substitute it with per gene-knock-in. <br />
<br><br><br />
This requires a further amplification step adding the specific “homology arms” which must match which must match the genomic DNA of the cell line being targeted, so in our case the native target gene sequence. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.<br />
<br><br><br />
It is the client’s responsibility to design and engineer the targeting construct. See our tips on targeting construct design. <br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasicTeam:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic2010-11-29T23:05:15Z<p>Georgio: New page: {{ESBS-Strasbourg/Temp2}} {| <html> <div style="position: absolute; width: 965px; height: 20px; z-index: 1; top: -40px; background-color:#222222" id="layer1"> <font color="#FFFFFF"> <im...</p>
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<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
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ProteOlux Plus</a></li><br />
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<div class="heading">ProteOlux Basic</div><br />
<div class="desc"><br />
<br />
<ul><br />
<li><a href="#Animation">Animation</a></li><br />
<li><a href="#molstruc">Molecular Structure of the System</a></li><br />
<li><a href="#molstrucunit">Molecular Structure of 1 unit</a></li><br />
<li><a href="#system">System description</a></li><br />
<li><a href="#assembly">Assembly</a></li><br />
<li><a href="#taggedprot">Tagged protein</a></li><br />
<li><a href="#mecadesc">Mechanical description</a></li><br />
<li><a href="#workstation">Workstation</a></li><br />
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<a name="degradation"></a><br />
Degradation system<br />
</div><br />
<br><br />
<p><b>Description</b></p><br />
ClpXP is an AAA protease present in bacteria, consisting of two main components, ClpX and ClpP. The ClpX is a hexamer consisting of six identical subunits. It recognizes specific degradation tags of target substrate proteins, unfolds them in an ATP-consuming hydrolysis reaction, and uses additional cycles of ATP hydrolysis to translocate the unfolded polypeptide into an interior chamber of ClpP, where proteolysis takes place. ClpP is a multi-subunit serine peptidase, in which the proteolytic active sites reside within a barrel-shaped structure. <br />
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<img src="https://static.igem.org/mediawiki/2010/d/d7/ESBS-Strasbourg-Clpp.png" width="220px" height="316px"><br />
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;<br />
<img src="https://static.igem.org/mediawiki/2010/e/e7/ESBS-Strasbourg-clpx.png" width="217px" height="316px"><br />
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<br><br><br />
In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. <br />
<br />
<br><br><br />
<p><b>The recognition sequence</b></p><br />
In the native organism, the SsrA tag is added to incomplete proteins whose translation has been aborted. Thus, misfunctionnal proteins do not accumulate inside the cell. The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. ClpX recognizes the last three residues LAA(Leu9, Ala10 and Ala11). Proteins with C-terminal LAA- tags are degraded rapidly in the cell, even without presence of SspB.<br />
<br><br />
The publication of Tania Baker <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[7]</a></i> based on the ClpXP protease of E. Coli which degrades substrates bearing the specific SsrA recognition sequence, has been the starting point of our reflection. In this work Baker and colleagues designed a series of modified ssrA tags which have weakened interactions with ClpXP to engineer controlled degradation. In E. coli, the adaptor SspB tethers ssrA-tagged substrates to the ClpXP protease, causing a modest increase in their rate of degradation. In the absence of SspB , substrates bearing the artificially altered DAS-tag were stable, in contrast the degradation of substrates bearing these engineered peptide tags was 100-fold more efficiently when SspB was present.<br />
<br><br><br />
To engineer controlled degradation, Baker and Sauer (2006) designed a series of modified SsrA tags that have weakened interactions with ClpXP. The DAS-tag presents one of these artificial sequence; its Kd value is significantly higher than the one of wild type SsrA, thus degradation of DAS-tagged proteins is not significant within the range of physiological concentrations. There is an N-terminal equivalent to the DAS-tag, the λO- tag. In the absence of SspB, substrates bearing the artificially altered tags are stable; however, through the action of the adaptor protein SspB, DAS- or λO –tagged proteins are significantly degraded.<br />
<br><br><br />
<p><b>Substitution of the adaptor</b></p><br />
The role of the adaptor-protein SspB has been assumed by Pif6 in our system, only light-induced activation can lead to binding and efficient degradation of DAS bearing constructs. <br />
<br />
<img src="https://static.igem.org/mediawiki/2010/8/82/ESBS-Strasbourg-ImageProteinfinal%2B.png" width="239px" height="168px" align="left"><br />
<p>&nbsp;</p><br />
<br />
<p><span>The target protein will be fused to Pif6 and to the specific degradation tag.<br />
<br>To avoid problems with the accessibility or an increased activity of the target protein we provide two different variants: the C-terminal degradation tags, with the target protein construct [PIF6-linker-Protein-DAS] and the N-terminally fused λO-tag resulting in the construct [λO-Protein-linker-PIF6]. </span></p><br />
<br />
<br />
<br><br><br />
<br><br><br />
For the C-terminal degradation tags, the target protein construct will be PIF-linker-Protein-Tag. To avoid problems with the accessibility of the tag we decided to test a further contruction with a N-terminally fused degradation tag (Tag-Protein-linker-PIF).<br />
<br><br />
We need then to choose an appropriate tag. It was also a critical step. <br />
<br><br><br />
<p><b>The LAA Tag</b></p><br />
The ssrA tag is a natural well-characterized recognition for ClpXP-degradation sequence in E. Coli. It is composed of the 11 amino acid sequence AANDENYALAA, localized at the C-terminal of the target protein. At least five ClpX-recognizing motifs have been determined: three located at the N-terminus and two at the C-terminus <i><a href="https://2010.igem.org/Team:ESBS-Strasbourg/Project/Reference">[34]</a></i>. Here we are concentrating on those located at the C-terminus where ClpX recognizes the last three residues (Leu9, Ala10 and Ala11).<br />
<br><br />
ClpX alone is able to interact with the ssrA-tagged substrates and delivers them to ClpP protease. However, it has been shown that the adaptor protein, SspB, markedly enhances the recognition of the ssrA tag. So, in our case the PIF-fused target should also favor the recognition of the ssrA tag.<br />
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<a name="molstruc"></a><br />
Molecular Structure of the System<br />
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<div class="desc"><br />
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<center><br />
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<img src="https://static.igem.org/mediawiki/2010/e/e7/ESBS-Strasbourg-Moleculare-structure.png"><br />
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<p><b>The left shows the molecular structure of the ClpX hexamere fused to the phytochrome B and the <br> schematic image in the animation</b></p><br />
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<a name="molstrucunit"></a><br />
Molecular structure of one unit (assembled biobrick) of our system.<br />
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<p><b>This shows the molecular structure of the trimetric unit of the ClpX fused to phytochrome B <br> and the schematic image in the animation.</b></p><br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteoluxTeam:ESBS-Strasbourg/proteolux2010-11-29T21:59:12Z<p>Georgio: </p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">ProteOlux</div><br />
<br><br />
<br><center><br />
<div style="position: relative; width: 800px; height: 40px; id="layer1" align="justify"><br />
You are interested in analyzing a specific protein in its natural cell environment? You want to analyze its dynamics, by installing any defined protein concentration in your cell without interfering with the cell’s metabolism? <b>Proteolux</b> offers the solution:</div></center><br />
<br><br />
<br />
<center><b>A specific light-controllable protein degradation system for any defined protein.</b></center><br />
<br><br><br />
<br />
<center><br />
<div style="position: relative; width: 800px; height: 80px; id="layer1" align="justify"><br />
Proteolux’s aim is to offer new and more sophisticated methods for protein analysis. Proteins are the actual active factors within cells and controlling protein concentrations leads to the control over the cell itself. Protein levels are installed by a complex homeostasis of de-novo protein synthesis and protein degradation. There are already some techniques for controlling specific protein expression. However, up to now, there are only a few systems that offer specific control about protein degradation. Most old techniques use protein knock-out or RNA interference to analyze the protein absence phenotype (figure 1).</div></center><br />
<br><br><br />
<br />
<center><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2010/d/d5/ESBS-Strasbourg-Prot-interference.png" width="500px" height="196px"></a><br />
</center><br />
<center><br />
<div style="position: relative; width: 500px; height: 60px; id="layer1" align="justify"><br />
<i><font color="#cccccc" size="1">Figure 1: Overview about the synthesis pathway of proteins and the different layers of regulation. The coding information of the DNA is transcribed into mRNA, which is further translated into proteins. The repression of expression protein can take place on three different layers of regulation. Classic methods include gene knockout or RNA interference, which contain several drawbacks. Proteolux defines a new layer of regulation directly on protein level, which can further regulate DNA and mRNA levels.</font></i></a></div><br />
</center><br />
<br><br><br />
<br />
<center><br />
<div style="position: relative; width: 800px; height: 350px; id="layer1" align="justify"><br />
Both methods exhibit several disadvantages. Gene knockout allows only the analysis of complete protein absence phenotypes, which creates problems in the case of essential proteins, whose absence is lethal to cells. Additionally, the creation of knock out strains (e.g. knock out mouse) for in-vivo analysis demands 3 generations and therefore in advance preparation of the experiment. RNA interference allows more specific regulation of protein expression than gene knockout. However, the main problems are the delivery of the RNAi, a design that is specific in order to avoid off-target effects. Moreover, the actual amount of protein in the cell cannot be controlled as the silencing percentage differs from interfering RNA to interfering RNA and it is difficult to achieve total silencing. Other disadvantages are the limited duration of the repression and toxic effects on cells.<br />
<br><br><br />
Proteolux’s specific light-controllable protein degradation system interacts directly on protein level, which allows complete control over the protein level as well as the over DNA and RNA layers. The Proteolux system offers the solution for the drawbacks of the other methods. The extremely high controllability allows fine regulation of any protein concentration, which allows also analysis of essential proteins. Different protein concentrations can be installed over time on demand of the experimenter. In-vivo experiments can be made directly after transfection with the system. Long preparation time before conducting experiments is not needed. The protein can be analyzed in its native conformation, as there is no tagging or fusion with other markers necessary. Furthermore, the metabolism of the cell is not altered, as the system is completely controlled by light. There is no off-target degradation due to the extreme high specificity of the interaction partners of the system. Moreover, the Proteolux system does not exhibit any toxicity to the cells. <br />
<br><br><br />
The light control of the Proteolux systems provides multiple advantages over the use of classical chemical inducers. The system responds instantly to the light signal, which can be used to specifically regulate its action. Chemical inducers do not respond immediately, due to the diffusion time to get to the place of action. Once the chemical inducer at its side of action it is not possible to turn the system off again. However, light offers this switchable control (on and off) of the system. Furthermore, light does not interact with the host metabolism which allows protein analysis in its unchanged natural environment. Another great advantage of light over chemical molecules is its inexpensiveness and its unlimited availability (summarized in the table below).<br />
</div></center><br />
<br><br />
<br />
<center><br />
<br><br />
<img src="https://static.igem.org/mediawiki/2010/e/ef/ESBS-Strasbourg-advantage.png" width="500px" height="209px"></a><br />
</center><br />
<br><br><br />
<center><br />
<b>We sparked your interest in Proteolux? Please follow the guide to learn more about the scientific background of our system.</b></center><br />
<br><br />
<center><br />
<a target="_blank" href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/guide"><br />
<img src="https://static.igem.org/mediawiki/2010/1/10/ESBS-Strasbourg-guide.gif" height="100px" width="82.4px"></a><br />
</center><br />
<center><b>Let me guide you !</b></center><br />
<br><br />
<br />
<br />
<br />
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</html></div>Georgiohttp://2010.igem.org/Team:ESBS-Strasbourg/proteoluxTeam:ESBS-Strasbourg/proteolux2010-11-29T21:57:33Z<p>Georgio: </p>
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;HOME&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview">Overview&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/overview/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
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<a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific"><br />
Scientific Background&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;|</a></p><br />
<ul><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxbasic"><br />
ProteOlux Basic</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxplus"><br />
ProteOlux Plus</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/scientific/proteoluxpro"><br />
ProteOlux Pro</a></li><br />
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<p><br/><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Application</a></p> <br />
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<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/cancer"><br />
Cancer</a></li><br />
<li><a href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/application/neuro"><br />
<font size="3">Neurodegenerative Diseases</font></a></li><br />
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<div class="heading">ProteOlux</div><br />
<br><br />
<br><center><br />
<div style="position: relative; width: 800px; height: 40px; id="layer1" align="justify"><br />
You are interested in analyzing a specific protein in its natural cell environment? You want to analyze its dynamics, by installing any defined protein concentration in your cell without interfering with the cell’s metabolism? <b>Proteolux</b> offers the solution:</div></center><br />
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<center><b>A specific light-controllable protein degradation system for any defined protein.</b></center><br />
<br><br><br />
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<center><br />
<div style="position: relative; width: 800px; height: 80px; id="layer1" align="justify"><br />
Proteolux’s aim is to offer new and more sophisticated methods for protein analysis. Proteins are the actual active factors within cells and controlling protein concentrations leads to the control over the cell itself. Protein levels are installed by a complex homeostasis of de-novo protein synthesis and protein degradation. There are already some techniques for controlling specific protein expression. However, up to now, there are only a few systems that offer specific control about protein degradation. Most old techniques use protein knock-out or RNA interference to analyze the protein absence phenotype (figure 1).</div></center><br />
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<img src="https://static.igem.org/mediawiki/2010/d/d5/ESBS-Strasbourg-Prot-interference.png" width="500px" height="196px"></a><br />
</center><br />
<center><br />
<div style="position: relative; width: 500px; height: 60px; id="layer1" align="justify"><br />
<i><font color="#cccccc" size="1">Figure 1: Overview about the synthesis pathway of proteins and the different layers of regulation. The coding information of the DNA is transcribed into mRNA, which is further translated into proteins. The repression of expression protein can take place on three different layers of regulation. Classic methods include gene knockout or RNA interference, which contain several drawbacks. Proteolux defines a new layer of regulation directly on protein level, which can further regulate DNA and mRNA levels.</font></i></a></div><br />
</center><br />
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<center><br />
<div style="position: relative; width: 800px; height: 350px; id="layer1" align="justify"><br />
Both methods exhibit several disadvantages. Gene knockout allows only the analysis of complete protein absence phenotypes, which creates problems in the case of essential proteins, whose absence is lethal to cells. Additionally, the creation of knock out strains (e.g. knock out mouse) for in-vivo analysis demands 3 generations and therefore in advance preparation of the experiment. RNA interference allows more specific regulation of protein expression than gene knockout. However, the main problems are the delivery of the RNAi, a design that is specific in order to avoid off-target effects. Moreover, the actual amount of protein in the cell cannot be controlled as the silencing percentage differs from interfering RNA to interfering RNA and it is difficult to achieve total silencing. Other disadvantages are the limited duration of the repression and toxic effects on cells.<br />
<br><br><br />
Proteolux’s specific light-controllable protein degradation system interacts directly on protein level, which allows complete control over the protein level as well as the over DNA and RNA layers. The Proteolux system offers the solution for the drawbacks of the other methods. The extremely high controllability allows fine regulation of any protein concentration, which allows also analysis of essential proteins. Different protein concentrations can be installed over time on demand of the experimenter. In-vivo experiments can be made directly after transfection with the system. Long preparation time before conducting experiments is not needed. The protein can be analyzed in its native conformation, as there is no tagging or fusion with other markers necessary. Furthermore, the metabolism of the cell is not altered, as the system is completely controlled by light. There is no off-target degradation due to the extreme high specificity of the interaction partners of the system. Moreover, the Proteolux system does not exhibit any toxicity to the cells. <br />
<br><br><br />
The light control of the Proteolux systems provides multiple advantages over the use of classical chemical inducers. The system responds instantly to the light signal, which can be used to specifically regulate its action. Chemical inducers do not respond immediately, due to the diffusion time to get to the place of action. Once the chemical inducer at its side of action it is not possible to turn the system off again. However, light offers this switchable control (on and off) of the system. Furthermore, light does not interact with the host metabolism which allows protein analysis in its unchanged natural environment. Another great advantage of light over chemical molecules is its inexpensiveness and its unlimited availability (summarized in the table below).<br />
</div></center><br />
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<br><br><br />
<center><br />
<b>We sparked your interest in Proteolux? Please follow the guide to learn more about the scientific background of our system.</b></center><br />
<br><br />
<center><br />
<a target="_blank" href="https://2010.igem.org/Team:ESBS-Strasbourg/proteolux/guide"><br />
<img src="https://static.igem.org/mediawiki/2010/1/10/ESBS-Strasbourg-guide.gif" height="100px" width="82.4px"></a><br />
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<center><b>Let me guide you !</b></center><br />
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