Team:Freiburg Bioware/Project/insertion of motifs into surface-exposed loops
From 2010.igem.org
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- | < | + | h1>His-Affinity tag</h1> |
- | <div align=center> | + | <div align="center"> |
- | + | <table class="MsoTableGrid" | |
- | <table class=MsoTableGrid | + | style="border: medium none ; margin-left: 180.8pt; border-collapse: collapse;" |
- | + | border="0" cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr style="height: 142.35pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 300.3pt; height: 142.35pt;" | |
- | + | valign="top" width="400"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img style="width: 395px; height: 198px;" | |
- | + | src="https://static.igem.org/mediawiki/2010/4/44/Freiburg10_His-tag.png" | |
- | + | alt="" align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 22.6pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 300.3pt; height: 22.6pt;" | |
- | + | valign="top" width="400"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span style="line-height: 200%;" lang="EN-US">Schematic | |
- | + | figure of His-affinity Tag insertion into the viral capsid</span></p> | |
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 18pt; line-height: 200%;" |
- | + | lang="EN-US"> </span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US">The insertion of a His-Affinity Tag |
- | lang=EN-US>The insertion of a His-Affinity Tag into the exposed major surface | + | into the exposed major surface |
loops of the viral vectors allows their specific affinity purification | loops of the viral vectors allows their specific affinity purification | ||
- | employing e.g. Ni-NTA affinity chromatography. This purification method was | + | employing e.g. Ni-NTA affinity chromatography. This purification method |
- | tested using the cell culture lysate of transfected AAV-293 cells that were | + | was |
- | either grown in DMEM supplemented with 10% FCS or in the serum-free GIBCO® | + | tested using the cell culture lysate of transfected AAV-293 cells that |
- | FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free media is | + | were |
- | a technological modification meant to facilitate the production of pure viral | + | either grown in DMEM supplemented with 10% FCS or in the serum-free |
- | vectors. Purified viral vectors are important for several applications such as | + | GIBCO® |
- | animal models and biophysical characterizations. On the other hand, the elution | + | FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free |
- | of the His-tagged viral vectors allows also enrichment of transgene viral | + | media is |
- | vectors. In order to answer the question if and to what degree viral vectors | + | a technological modification meant to facilitate the production of pure |
- | are transferred into the media, the cells were centrifugated, then divided into | + | viral |
- | the pellet fraction and the supernatant. Physical cell lysis was performed for | + | vectors. Purified viral vectors are important for several applications |
- | both fractions of the two produced batches (serum-free and FCS media) by | + | such as |
+ | animal models and biophysical characterizations. On the other hand, the | ||
+ | elution | ||
+ | of the His-tagged viral vectors allows also enrichment of transgene | ||
+ | viral | ||
+ | vectors. In order to answer the question if and to what degree viral | ||
+ | vectors | ||
+ | are transferred into the media, the cells were centrifugated, then | ||
+ | divided into | ||
+ | the pellet fraction and the supernatant. Physical cell lysis was | ||
+ | performed for | ||
+ | both fractions of the two produced batches (serum-free and FCS media) | ||
+ | by | ||
performing four cycles of freeze and thaw.</span></p> | performing four cycles of freeze and thaw.</span></p> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | <table class=MsoTableGrid | + | cellpadding="0" cellspacing="0"> |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img style="width: 627px; height: 567px;" alt="" | |
- | + | id="Bild 1" | |
- | + | src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_His-experiment.png"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">Experimental setup for the | |
- | + | purification of His-tag presenting viral particles produced by AAV-293 | |
- | + | cells in DMEM or the protein- and animal-free Freestyle medium. | |
- | + | Purified viral particles can be detected by ELISA using an | |
+ | anti-poly-Histidine antibody or qPCR for encapsidated vector plasmids, | ||
+ | respectively</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US"> | |
- | <p class=MsoNormal | + | <h2>Material and Methods:</h2> |
- | lang=EN-US>Material and Methods:</span></p> | + | </span></p> |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US">Transfection of the AAV-293 producer |
- | lang=EN-US>Transfection of the AAV-293 producer cells was performed in five 10 cm | + | cells was performed in five 10 cm |
- | petri dishes with 3.6x10^6 cells, resulting in a confluency of about 70-80% | + | petri dishes with 3.6x10^6 cells, resulting in a confluency of about |
- | according to the standard protocol either with cells grown in GIBCO® FreeStyle™ | + | 70-80% |
- | 293 Expression Medium (Invitrogen, protein- and animal-origin free) or in DMEM | + | according to the standard protocol either with cells grown in GIBCO® |
+ | FreeStyle™ | ||
+ | 293 Expression Medium (Invitrogen, protein- and animal-origin free) or | ||
+ | in DMEM | ||
supplemented with 10% FCS (PAA). For transfection, the composite parts | supplemented with 10% FCS (PAA). For transfection, the composite parts | ||
- | pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an 1:1 ratio | + | pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an |
- | together with </span><span | + | 1:1 ratio |
- | and <span class=apple-style-span><span style= | + | together with </span><span style="font-size: 12pt; line-height: 200%;" |
- | lang=EN-US> Cells were spun down at 200 x g for five minutes and the samples | + | lang="EN-US">pHelper |
- | were divided into the pellet and the supernatant fractions. Physical cell lysis | + | and <span class="apple-style-span"><span style="color: black;">[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR.</span></span></span><span |
- | was performed by four cycles of freeze and thaw for all four samples. The cell | + | lang="EN-US"> Cells were spun down at 200 x g for five minutes and the |
- | lysate / supernatant fractions were incubated with 800 µl of His-Affinity Gel | + | samples |
- | (kindly provided by Zymo Research, USA) at 4 °C for 18 hours with 200 rpm constant | + | were divided into the pellet and the supernatant fractions. Physical |
- | agitation. The beads were then collected in 5 ml gravity-flow columns and | + | cell lysis |
- | washed five times with one column volume of PBS each. The His-affinity gel was subsequently | + | was performed by four cycles of freeze and thaw for all four samples. |
- | washed with PBS, 25 mM Imidazole to remove unspecifically bound proteins. | + | The cell |
- | Elution was performed in an second step with PBS, 500 mM Imidazole to elute the | + | lysate / supernatant fractions were incubated with 800 µl of |
- | His-tagged viral vectors. The genomic titer of the purified viral vectors was | + | His-Affinity Gel |
- | detected via q-PCR. In an ELISA, viral vectors were captured employing the | + | (kindly provided by Zymo Research, USA) at 4 °C for 18 hours with 200 |
- | monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, DKFZ, Heidelberg) | + | rpm constant |
+ | agitation. The beads were then collected in 5 ml gravity-flow columns | ||
+ | and | ||
+ | washed five times with one column volume of PBS each. The His-affinity | ||
+ | gel was subsequently | ||
+ | washed with PBS, 25 mM Imidazole to remove unspecifically bound | ||
+ | proteins. | ||
+ | Elution was performed in an second step with PBS, 500 mM Imidazole to | ||
+ | elute the | ||
+ | His-tagged viral vectors. The genomic titer of the purified viral | ||
+ | vectors was | ||
+ | detected via q-PCR. In an ELISA, viral vectors were captured employing | ||
+ | the | ||
+ | monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, | ||
+ | DKFZ, Heidelberg) | ||
that exclusively recognizes assembled AAV capsids. His-Tags present in | that exclusively recognizes assembled AAV capsids. His-Tags present in | ||
- | assembled viral capsids were subsequently detected with an HRP-tagged secondary | + | assembled viral capsids were subsequently detected with an HRP-tagged |
- | anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). </span><span | + | secondary |
- | style= | + | anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). </span><span |
- | peroxidase substrate ABTS. Generation of blue-green color (absorption at 405 | + | style="font-size: 12pt; line-height: 200%;" lang="EN-US">HRP presence |
- | nm) was measured in a Tecan Sunrise plate reader. Sample data were blanked with | + | was detected using the |
+ | peroxidase substrate ABTS. Generation of blue-green color (absorption | ||
+ | at 405 | ||
+ | nm) was measured in a Tecan Sunrise plate reader. Sample data were | ||
+ | blanked with | ||
the average of the non-template controls (NTC).</span></p> | the average of the non-template controls (NTC).</span></p> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US"> | |
- | <p class=MsoNormal | + | <h2>Results and Discussion</h2> |
- | lang=EN-US>Results and Discussion | + | </span></p> |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span | |
- | + | lang="EN-US">Presence of the His-affinity tag in | |
- | + | the viral capsid was detected and the ELISA enabled quantification of | |
- | + | the | |
- | + | purification procedure efficiency. The absorbance measured for the | |
- | + | elution | |
- | + | fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and | |
- | + | 0.5 for | |
- | + | the Free Style 293-grown cells, assigning the DMEM-grown cells a five | |
- | + | times | |
- | + | higher production efficiency. Comparison between the cell pellet and | |
- | <p class=MsoNormal style= | + | the |
- | 0cm;line-height:normal | + | supernatant fractions revealed that 70 - 80% of the viral particles can |
- | the viral capsid was detected and the ELISA enabled quantification of the | + | be |
- | purification procedure efficiency. The absorbance measured for the elution | + | |
- | fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and 0.5 for | + | |
- | the Free Style 293-grown cells, assigning the DMEM-grown cells a five times | + | |
- | higher production efficiency. Comparison between the cell pellet and the | + | |
- | supernatant fractions revealed that 70 - 80% of the viral particles can be | + | |
found inside the producer cells.</span></p> | found inside the producer cells.</span></p> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | + | align="left"><span lang="EN-US">According to these results, | |
- | + | producer cells should be grown in complex media for <i>in vitro</i> | |
- | + | and cell | |
- | text-indent:0cm;line-height:115% | + | culture experiments. Use of serum-free produced viral vectors is |
- | producer cells should be grown in complex media for <i>in vitro</i> and cell | + | recommended |
- | culture experiments. Use of serum-free produced viral vectors is recommended | + | for mouse or other animal experiments and possible therapeutical |
- | for mouse or other animal experiments and possible therapeutical applications | + | applications |
where even the presence of traces amounts of fetal calf serum should be | where even the presence of traces amounts of fetal calf serum should be | ||
avoided. Combination with different purification approaches such as gel | avoided. Combination with different purification approaches such as gel | ||
- | filtration chromatography using i.e. Superdex 200 columns (GE Healthcare) | + | filtration chromatography using i.e. Superdex 200 columns (GE |
- | enables the production of highly purified viral vectors for several different | + | Healthcare) |
- | applications.<br | + | enables the production of highly purified viral vectors for several |
+ | different | ||
+ | applications.<br style="page-break-before: always;" clear="all"> | ||
</span></p> | </span></p> | ||
- | + | <table class="MsoTableGrid" | |
- | <table class=MsoTableGrid | + | style="border: medium none ; border-collapse: collapse;" border="0" |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><img style="width: 451px; height: 796px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/0/0d/Freiburg10_His-data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><span lang="EN-US">A: Schematic overview of the sandwich | |
- | + | ELISA for the detection of His-tagged viral particles</span></p> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
- | + | align="left"><span lang="EN-US">B: ELISA from viral particles produced | |
- | + | by AAV-293 cells in DMEM or Free Style medium, divided into cell pellet | |
- | + | and cell culture supernatant samples. The particles were purified using | |
- | + | Ni-NTA affinity chromatography with Imidazole in PBS as washing and | |
- | + | elution agent</span></p> | |
- | + | <p class="MsoNormal" | |
- | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | |
+ | align="left"><span lang="EN-US">C: Absorption measurements from plate | ||
+ | shown in B. Undiluted Äkta fractions converted ABTS peroxidase | ||
+ | substrate at 405 nm</span></p> | ||
+ | <p class="MsoNormal" | ||
+ | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" | ||
+ | align="left"><span lang="EN-US">D: As C, whereas Äkta fractions were | ||
+ | 10-fold diluted</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <span | |
- | <span | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";" |
- | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> | |
</span> | </span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span lang="EN-US"> </span></p> |
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span lang="EN-US"> </span></p> |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 20pt; line-height: 200%;" |
- | + | lang="EN-US"> | |
- | Peptide (BAP)</span></p> | + | <h1>Biotinylation Acceptor |
- | + | Peptide (BAP)</h1> | |
- | <div align=center> | + | </span></p> |
- | + | <div align="center"> | |
- | <table class=MsoTableGrid | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr style="height: 187.45pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 327.6pt; height: 187.45pt;" | |
- | + | valign="top" width="437"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | + | style="width: 419px; height: 244px;" | |
- | + | src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_BAP-tag.png" | |
- | + | alt="http://partsregistry.org/wiki/images/a/a9/Freiburg10_ViralBrick_motif_BAP.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 39.75pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 327.6pt; height: 39.75pt;" | |
- | + | valign="top" width="437"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
+ | style="line-height: 200%;" lang="EN-US">Schematic overview of BAP | ||
+ | insertion into AAV-2 viral capsids, followed by biotinylation and | ||
+ | binding of streptavidin-coupled molecules</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">The | |
- | <p class=MsoNormal style= | + | BAP |
- | (Biotinylation Acceptor Peptide) included in the Virus Construction Kit is a 15 | + | (Biotinylation Acceptor Peptide) included in the Virus Construction Kit |
- | amino acid peptide identified by Schatz et al. (1993) in a library screening | + | is a 15 |
- | approach and published under the number #85. This peptide with the sequence | + | amino acid peptide identified by Schatz et al. (1993) in a library |
- | GLNDIFEAQKIEWHE contains a central lysine residue that can be specifically | + | screening |
- | biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded by the | + | approach and published under the number #85. This peptide with the |
- | BirA gene of <i>E. coli</i>. Specific biotinylation of this peptide sequence | + | sequence |
- | can be performed in vivo by cotransfecting a plasmid with the BirA gene as | + | GLNDIFEAQKIEWHE contains a central lysine residue that can be |
- | described for the AAV by Arnold et al. (2006) or by an in vitro coupling | + | specifically |
- | approach using the purified Escherichia coli enzyme biotin ligase (BirA). </span></p> | + | biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded |
- | + | by the | |
- | <p class=MsoNormal style= | + | BirA gene of <i>E. coli</i>. Specific biotinylation of this peptide |
- | specifically coupled to the viral loops can either be used to attach Streptavidin-coupled | + | sequence |
- | molecules to the viral capsid or to chemically couple molecules with a reactive | + | can be performed in vivo by cotransfecting a plasmid with the BirA gene |
- | group to biotin. Consequently, the inserted motif can enable the visualization | + | as |
+ | described for the AAV by Arnold et al. (2006) or by an in vitro | ||
+ | coupling | ||
+ | approach using the purified Escherichia coli enzyme biotin ligase | ||
+ | (BirA). </span></p> | ||
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Biotin | ||
+ | molecules | ||
+ | specifically coupled to the viral loops can either be used to attach | ||
+ | Streptavidin-coupled | ||
+ | molecules to the viral capsid or to chemically couple molecules with a | ||
+ | reactive | ||
+ | group to biotin. Consequently, the inserted motif can enable the | ||
+ | visualization | ||
of single virus particles by coupling fluorophores to the virus capsid | of single virus particles by coupling fluorophores to the virus capsid | ||
empowering further uses of the Virus Construction Kit in fundamental | empowering further uses of the Virus Construction Kit in fundamental | ||
Line 240: | Line 303: | ||
Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or | Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or | ||
Affibodies) can be coupled for manifold targeting approaches.</span></p> | Affibodies) can be coupled for manifold targeting approaches.</span></p> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | |
- | <p class=MsoNormal style= | + | <span |
- | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";" | |
- | <span | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> |
- | + | ||
</span> | </span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span lang="EN-US"> </span></p> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> | |
- | <p class=MsoNormal style= | + | <h2>Materials |
- | + | and Methods:</h2> | |
- | <p class=MsoNormal style= | + | </span></p> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US"> </span></p> | |
- | <p class=MsoNormal style= | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">Transfection |
- | AAV-293 producer cells was performed in five 10 cm petri dishes with 3.4x10^6 | + | of the |
- | cells (grown in DMEM supplemented with 10% FCS) resulting in a confluence of | + | AAV-293 producer cells was performed in five 10 cm petri dishes with |
- | 70-80% according to the standard protocol. Producer cells were harvested and | + | 3.4x10^6 |
- | together with the culture supernatant subjected to four cycles of freeze and | + | cells (grown in DMEM supplemented with 10% FCS) resulting in a |
- | thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 minutes and | + | confluence of |
- | concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight cut-off to | + | 70-80% according to the standard protocol. Producer cells were |
- | yield two milliliters. Genomic DNA attached to the virus particles was degraded | + | harvested and |
- | using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for one | + | together with the culture supernatant subjected to four cycles of |
- | hour. The concentrated cell lysate was washed three times with 4 ml 20 mM | + | freeze and |
- | bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500 µl were | + | thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 |
- | loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex 200 gel | + | minutes and |
- | filtration column (GE Healthcare). This purification is also described by <span | + | concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight |
- | class=apple-converted-space><span style= | + | cut-off to |
- | around the void volume giving a UV absorbance peak were pooled and applied to a | + | yield two milliliters. Genomic DNA attached to the virus particles was |
- | Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading volume) and | + | degraded |
- | concentrated to 500 µl. This purified virus sample was washed four times with 10 | + | using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for |
- | mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin ligase | + | one |
+ | hour. The concentrated cell lysate was washed three times with 4 ml 20 | ||
+ | mM | ||
+ | bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500 | ||
+ | µl were | ||
+ | loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex | ||
+ | 200 gel | ||
+ | filtration column (GE Healthcare). This purification is also described | ||
+ | by <span class="apple-converted-space"><span style="color: black;">Smith | ||
+ | et al. (2003). Fractions | ||
+ | around the void volume giving a UV absorbance peak were pooled and | ||
+ | applied to a | ||
+ | Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading | ||
+ | volume) and | ||
+ | concentrated to 500 µl. This purified virus sample was washed four | ||
+ | times with 10 | ||
+ | mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin | ||
+ | ligase | ||
that was kindly provided by Avidity LCC (Colorado, USA).</span></span></span></p> | that was kindly provided by Avidity LCC (Colorado, USA).</span></span></span></p> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal style= | + | class="apple-converted-space"><span style="color: black;" lang="EN-US">The |
- | + | BAP-containing viral vector sample (500 µl) | |
- | was mixed according to the manufacturer protocol with each 72 µl Biomix A, | + | was mixed according to the manufacturer protocol with each 72 µl Biomix |
+ | A, | ||
Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl | Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl | ||
- | containing 25000 units of the biotin ligase BirA was added to the reaction | + | containing 25000 units of the biotin ligase BirA was added to the |
- | mixture and incubated for 6 h at 30 °C. In order to remove unbound biotin, the | + | reaction |
- | biotinylated viral vectors were washed five times with 10 mM Tris buffer pH 8.0. | + | mixture and incubated for 6 h at 30 °C. In order to remove unbound |
+ | biotin, the | ||
+ | biotinylated viral vectors were washed five times with 10 mM Tris | ||
+ | buffer pH 8.0. | ||
</span></span></p> | </span></span></p> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal style= | + | class="apple-converted-space"><span style="color: black;" lang="EN-US"> </span></span></p> |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | class="apple-converted-space"><span style="color: black;" lang="EN-US">Biotinylation | |
- | <p class=MsoNormal style= | + | was verified with an ELISA as |
- | + | depicted in figure A. For the detection of successfully biotinylated | |
- | depicted in figure A. For the detection of successfully biotinylated viral | + | viral |
- | vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng monoclonal A20 | + | vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng |
- | antibody per well for eight hours at 4°C, and blocked over night with PBST + | + | monoclonal A20 |
- | 0.5 % BSA. This plate was incubated for 1 h at room temperature with 100 µl of | + | antibody per well for eight hours at 4°C, and blocked over night with |
+ | PBST + | ||
+ | 0.5 % BSA. This plate was incubated for 1 h at room temperature with | ||
+ | 100 µl of | ||
serial diluted viral vectors. After washing the plates three times, the | serial diluted viral vectors. After washing the plates three times, the | ||
- | recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was added to | + | recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was |
- | each well for 1 h at room temperature. Again, the plates were washed three | + | added to |
- | times followed by detection of absorbance caused by converted ABTS substrate at | + | each well for 1 h at room temperature. Again, the plates were washed |
+ | three | ||
+ | times followed by detection of absorbance caused by converted ABTS | ||
+ | substrate at | ||
405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.</span></span></p> | 405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.</span></span></p> | ||
- | + | <span class="apple-converted-space"><span | |
- | <span class=apple-converted-space><span | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif"; color: black;" |
- | line-height:115%;font-family: | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> |
- | style= | + | |
</span></span> | </span></span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span class="apple-converted-space"><span |
- | + | style="color: black;" lang="EN-US"> </span></span></p> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal style= | + | class="apple-converted-space"><span style="color: black;" lang="EN-US"> </span></span></p> |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | <table class=MsoTableGrid | + | cellpadding="0" cellspacing="0"> |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | + | style="width: 587px; height: 539px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/2/2c/Freiburg10_BAP-data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | class="apple-converted-space"><span style="color: black;" lang="EN-US">A: | |
- | + | Schematic overview of the detection of BAP-presenting viral particles | |
- | + | immobilized by A20 capture antibodies. Biotinylated capsids can be | |
- | + | detected by streptavidin-coupled HRP</span></span></p> | |
+ | <p class="MsoNormal" style="text-indent: 0cm;"><span | ||
+ | class="apple-converted-space"><span style="color: black;" lang="EN-US">B: | ||
+ | ELISA AAV-2 particles carrying a BAP insertion in loop 587 which can be | ||
+ | specifically biotinylated in vitro</span></span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal style= | + | class="apple-converted-space"><span style="color: black;" lang="EN-US"> </span></span></p> |
- | + | <span class="apple-converted-space"><span | |
- | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif"; color: black;" | |
- | <span class=apple-converted-space><span | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> |
- | line-height:115%;font-family: | + | |
- | style= | + | |
</span></span> | </span></span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span class="apple-converted-space"><span |
- | + | style="color: black;" lang="EN-US"> </span></span></p> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | <p class=MsoNormal style= | + | class="apple-converted-space"><span style="color: black;" lang="EN-US"> |
- | + | <h2>Results | |
- | lang=EN-US> </span></p> | + | and Discussion:</h2> |
- | + | </span></span><span lang="EN-US"> </span></p> | |
- | <p class=MsoNormal style= | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">As |
- | figure B, biotinylation of assembled AAV particles was achieved with the | + | can be seen in |
- | dilution series ranging from 2- to 128-fold. Correlating this assay with the | + | figure B, biotinylation of assembled AAV particles was achieved with |
- | qPCR experiment for the detection of encapsidated vector plasmid (genomic titer | + | the |
+ | dilution series ranging from 2- to 128-fold. Correlating this assay | ||
+ | with the | ||
+ | qPCR experiment for the detection of encapsidated vector plasmid | ||
+ | (genomic titer | ||
using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant | using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant | ||
- | particles (DRP). Consequently, the presence of approximately 3.6x10^5 biotinylated | + | particles (DRP). Consequently, the presence of approximately 3.6x10^5 |
- | DRPs can be detected using the described ELISA. Compared to the virus particle | + | biotinylated |
- | detection ELISA emploing A20 as capture- and detection antibody, the detection | + | DRPs can be detected using the described ELISA. Compared to the virus |
- | limit of the biotinylated viral capsids is about 10fold more sensitive. This | + | particle |
- | may be explained by the higher affinity of strepavidin towards biotin relative | + | detection ELISA emploing A20 as capture- and detection antibody, the |
- | to the A20 antibody affinity or multiple biotinylation of a single virus | + | detection |
+ | limit of the biotinylated viral capsids is about 10fold more sensitive. | ||
+ | This | ||
+ | may be explained by the higher affinity of strepavidin towards biotin | ||
+ | relative | ||
+ | to the A20 antibody affinity or multiple biotinylation of a single | ||
+ | virus | ||
particle.</span></p> | particle.</span></p> | ||
- | + | <span | |
- | <span | + | style="font-size: 20pt; line-height: 115%; font-family: "Calibri","sans-serif";" |
- | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> | |
</span> | </span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span style="font-size: 20pt; line-height: 115%;" |
- | line-height:115% | + | lang="EN-US"> </span></p> |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 20pt; line-height: 200%;" |
- | + | lang="EN-US"> | |
- | binding domain (Z34C)</span></p> | + | <h1>Miniaturized antibody |
- | + | binding domain (Z34C)</h1> | |
- | <table class=MsoTableGrid | + | </span></p> |
- | + | <table class="MsoTableGrid" | |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr style="height: 184.6pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt; height: 184.6pt;" | |
- | + | valign="top" width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><img | |
- | + | style="width: 451px; height: 225px;" | |
- | + | src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Z34C-tag.png" | |
- | + | alt="" align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 23.7pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt; height: 23.7pt;" | |
- | + | valign="top" width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
+ | style="line-height: 200%;" lang="EN-US">Schematic overview of Z34C | ||
+ | insertion into AAV-2 viral capsids enabling antibody arming for therapy</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">The | |
- | <p class=MsoNormal style= | + | idea of this |
targeting approach is to utilize a minimized fragment of the </span><a | targeting approach is to utilize a minimized fragment of the </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/protein/153106"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/153106"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Staphylococcal |
- | lang=EN-US> that was first described in Staphylococcus aureus. These | + | Protein A</span></a><span lang="EN-US"> that was first described |
- | gram-positive bacteria have evolved the 508 amino acid long protein A that has | + | in Staphylococcus aureus. These |
- | a high affinity for the Fc-domain of antibodies to protect itself from the | + | gram-positive bacteria have evolved the 508 amino acid long protein A |
- | immune system. Binding to the constant region of the antibodies is accomplished | + | that has |
+ | a high affinity for the Fc-domain of antibodies to protect itself from | ||
+ | the | ||
+ | immune system. Binding to the constant region of the antibodies is | ||
+ | accomplished | ||
by the </span><a href="http://www.ncbi.nlm.nih.gov/protein/1Q2NA"><span | by the </span><a href="http://www.ncbi.nlm.nih.gov/protein/1Q2NA"><span | ||
- | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z-Domain</span></a><span | |
- | lang=EN-US> of Protein A that is 58-59 amino acids long, has alone a high | + | lang="EN-US"> of Protein A that is 58-59 amino acids long, has |
- | affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle structure. | + | alone a high |
+ | affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle | ||
+ | structure. | ||
In </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/8650153"><span | In </span><a href="http://www.ncbi.nlm.nih.gov/pubmed/8650153"><span | ||
- | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Braisted | |
- | 1996]</span></a><span lang=EN-US> the authors reduced the secundary | + | & Wells; |
- | structure to an two-helix bundle. This size reduction has lead to an drastic | + | 1996]</span></a><span lang="EN-US"> the authors reduced the |
- | reduction of the affinity for IgG (>10^5 fold) which could be recovered by | + | secundary |
- | 13 amino acid exchanges resulting in a 38 amino acid long peptide with an | + | structure to an two-helix bundle. This size reduction has lead to an |
+ | drastic | ||
+ | reduction of the affinity for IgG (>10^5 fold) which could be | ||
+ | recovered by | ||
+ | 13 amino acid exchanges resulting in a 38 amino acid long peptide with | ||
+ | an | ||
satisfying affinity for IgG (Kd = 185 nM) termed </span><a | satisfying affinity for IgG (Kd = 185 nM) termed </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDAA"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/1ZDAA"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z38</span></a><span |
+ | lang="EN-US">. | ||
This binding domain was subsequently improved in </span><a | This binding domain was subsequently improved in </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/pubmed/9294166"><span | + | href="http://www.ncbi.nlm.nih.gov/pubmed/9294166"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Starovasnik |
- | lang=EN-US> by the insertion of a disulfide bridge connecting the ends of | + | et al.; 1997]</span></a><span lang="EN-US"> by the insertion of a |
+ | disulfide bridge connecting the ends of | ||
the helices leading to the binding domain </span><a | the helices leading to the binding domain </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z34C</span></a><span |
+ | lang="EN-US"> which | ||
shows an increased affinity for IgG (Kd = 20 nM).</span></p> | shows an increased affinity for IgG (Kd = 20 nM).</span></p> | ||
- | + | <p class="MsoNormal"><span | |
- | <p class=MsoNormal><span | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" |
- | font-family: | + | lang="EN-US"> </span></p> |
- | + | <table class="MsoTableGrid" | |
- | <table class=MsoTableGrid | + | style="border: medium none ; border-collapse: collapse;" border="0" |
- | + | cellpadding="0" cellspacing="0"> | |
- | + | <tbody> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;"><img | |
- | + | style="border: 0px solid ; width: 524px; height: 309px;" id="Grafik 30" | |
- | + | src="https://static.igem.org/mediawiki/2010/c/cd/Freiburg10_Z34C-history.png" | |
- | + | alt=""></span></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-indent: 0cm;"><span | |
- | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" | |
+ | lang="EN-US">Development of the Z34C motif, a miniaturized antibody | ||
+ | binding motif</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal"><span | |
- | <p class=MsoNormal><span | + | style="font-size: 10pt; line-height: 200%; font-family: "Arial","sans-serif"; color: black;" |
- | font-family: | + | lang="EN-US"><br> |
- | </span><span lang=EN-US>This engineered antibody binding domain of 34 amino | + | </span><span lang="EN-US">This engineered antibody binding domain of 34 |
- | acids was then inserted into capsids of different viral vectors amongst others | + | amino |
+ | acids was then inserted into capsids of different viral vectors amongst | ||
+ | others | ||
also the AAV. In </span><a | also the AAV. In </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155067"><span | + | href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155067"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Ried |
- | lang=EN-US> the </span><a | + | et al.; 2002]</span></a><span lang="EN-US"> the </span><a |
- | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span | + | href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">Z34C</span></a><span |
- | was inserted at position 587 into the capsid of the AAV resulting in viral | + | lang="EN-US"> domain |
+ | was inserted at position 587 into the capsid of the AAV resulting in | ||
+ | viral | ||
vector that can be targeted to different target cells without genetic | vector that can be targeted to different target cells without genetic | ||
engineering. This targeting approach was then improved in </span><a | engineering. This targeting approach was then improved in </span><a | ||
- | href="http://www.ncbi.nlm.nih.gov/pubmed/15922956"><span | + | href="http://www.ncbi.nlm.nih.gov/pubmed/15922956"><span |
- | style= | + | style="color: windowtext; text-decoration: none;" lang="EN-US">[Gigout |
- | lang=EN-US> by the creation of mosaic vectors that contain only ~25% of | + | et al.; 2005]</span></a><span lang="EN-US"> by the creation of |
- | recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude more | + | mosaic vectors that contain only ~25% of |
+ | recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude | ||
+ | more | ||
infectiosity compared to all-mutant viruses.</span></p> | infectiosity compared to all-mutant viruses.</span></p> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 12pt; line-height: 200%;" |
- | + | lang="EN-US"> </span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 14pt; line-height: 200%;" |
- | + | lang="EN-US"> | |
- | + | <h2>Material and Methods:</h2> | |
- | <p class=MsoNormal | + | </span></p> |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | AAV-293 producer cells was performed for three different loop insertions of | + | align="left"><span style="font-size: 12pt; line-height: 200%;" |
- | Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in a | + | lang="EN-US">Transfection of the |
- | confluence of about 70-80%. For the transfection, either the composite parts pCMV_VP123(453-Z34C), | + | AAV-293 producer cells was performed for three different loop |
- | pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were cotransfected | + | insertions of |
- | with pHelper, <span class=apple-style-span><span style= | + | Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in |
- | and RepVP</span></span>123(587KO). The producer cells were harvested with the | + | a |
- | culture supernatant and subjected to four cycles of freeze and thaw cell lysis. | + | confluence of about 70-80%. For the transfection, either the composite |
- | The cell lysate was centrifuged at 4000 rpm for 15 minutes and concentrated in | + | parts pCMV_VP123(453-Z34C), |
- | a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight cut-off to | + | pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were |
- | two milliliters and washed three to five times with 20 mM Bis-Tris buffer, pH 6. | + | cotransfected |
- | The cell lysates were incubated with 250 units of Benzonase (Sigma-Aldrich) to | + | with pHelper, <span class="apple-style-span"><span |
- | remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE Healthcare) equipped | + | style="color: black;">[AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR |
- | with a Superdex 200 gel filtration column (GE Healthcare). Fractions (500 µl) | + | and RepVP</span></span>123(587KO). The producer cells were harvested |
- | around the void volume containing viral particles were collected and used in a | + | with the |
- | sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab (Imclone/Merck/Bristol-Myers | + | culture supernatant and subjected to four cycles of freeze and thaw |
- | Squibb). Detection was performed using the monoclonal antibody A20 (kindly | + | cell lysis. |
- | provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was biotinylated | + | The cell lysate was centrifuged at 4000 rpm for 15 minutes and |
- | using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP (Sigma-Aldrich). | + | concentrated in |
- | The HRP presence was detected by the conversion of the substrate ABTS at 405 | + | a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight |
- | nm. The average of the non-template controls (NTC) was subtracted from the | + | cut-off to |
+ | two milliliters and washed three to five times with 20 mM Bis-Tris | ||
+ | buffer, pH 6. | ||
+ | The cell lysates were incubated with 250 units of Benzonase | ||
+ | (Sigma-Aldrich) to | ||
+ | remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE | ||
+ | Healthcare) equipped | ||
+ | with a Superdex 200 gel filtration column (GE Healthcare). Fractions | ||
+ | (500 µl) | ||
+ | around the void volume containing viral particles were collected and | ||
+ | used in a | ||
+ | sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab | ||
+ | (Imclone/Merck/Bristol-Myers | ||
+ | Squibb). Detection was performed using the monoclonal antibody A20 | ||
+ | (kindly | ||
+ | provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was | ||
+ | biotinylated | ||
+ | using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP | ||
+ | (Sigma-Aldrich). | ||
+ | The HRP presence was detected by the conversion of the substrate ABTS | ||
+ | at 405 | ||
+ | nm. The average of the non-template controls (NTC) was subtracted from | ||
+ | the | ||
sample data.</span></p> | sample data.</span></p> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span style="font-size: 12pt; line-height: 200%;" |
- | + | lang="EN-US"> </span></p> | |
- | + | <span | |
- | <span | + | style="font-size: 11pt; line-height: 115%; font-family: "Calibri","sans-serif";" |
- | + | lang="EN-US"><br style="page-break-before: always;" clear="all"> | |
</span> | </span> | ||
- | + | <p class="MsoNormal" | |
- | <p class=MsoNormal | + | style="margin-bottom: 10pt; text-align: left; text-indent: 0cm; line-height: 115%;" |
- | text-indent:0cm;line-height:115% | + | align="left"><span lang="EN-US"> </span></p> |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> |
- | lang=EN-US>Results and Discussion:</span></p> | + | <h2>Results and Discussion:</h2> |
- | + | </span></p> | |
- | <div align=center> | + | <div align="center"> |
- | + | <table class="MsoTableGrid" | |
- | <table class=MsoTableGrid | + | style="border: medium none ; width: 384.6pt; border-collapse: collapse;" |
- | + | border="0" cellpadding="0" cellspacing="0" width="513"> | |
- | + | <tbody> | |
- | + | <tr style="height: 305.8pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 384.6pt; height: 305.8pt;" | |
- | + | valign="top" width="513"> | |
- | + | <p class="MsoNormal" style="text-align: center; text-indent: 0cm;" | |
- | + | align="center"><img style="width: 421px; height: 722px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_Z34C_affinity_data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 15.45pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 384.6pt; height: 15.45pt;" | |
- | + | valign="top" width="513"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">A: Sandwich-ELISA scheme for the | |
- | + | detection of Z34C-presenting viral particles. Immobilization is | |
- | + | achieved by binding a IgG molecule. Intact viral capsids can be | |
- | + | specifically bound by the biotinylated A20 antibody which can be | |
- | + | detected by Strepavidin-HRP</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">B: ELISA using the principle described | |
- | + | in A for loop insertion samples obtained by gel filtration | |
- | + | chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold | |
- | + | diluted samples were employed</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">C: ELISA signals obtained by | |
- | + | absorbance measurements from B at 405 nm, undiluted samples</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
+ | align="left"><span lang="EN-US">D: ELISA signals obtained by | ||
+ | absorbance measurements from B at 405 nm, diluted samples</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US">Three samples of different viral | |
- | <p class=MsoNormal | + | particles with insertions of the |
- | lang=EN-US>Three samples of different viral particles with insertions of the | + | antibody-binding motif Z34C were successfully purified by the gel |
- | antibody-binding motif Z34C were successfully purified by the gel filtration | + | filtration |
- | chromatography. The fractions around the void volume were subsequently used in | + | chromatography. The fractions around the void volume were subsequently |
- | two different sandwich ELISAs. The first one aims at the detection of the | + | used in |
- | particle’s ability to bind IgG-antibodies. The therapeutical antibody Cetuximab | + | two different sandwich ELISAs. The first one aims at the detection of |
- | was employed to test the affinity of the virus particles. As displayed in | + | the |
- | figure 1A, only Z34C-presenting particles should be immobilized and only | + | particle’s ability to bind IgG-antibodies. The therapeutical antibody |
- | assembled viral capsid will be detected due to the affinity of the A20 antibody | + | Cetuximab |
- | (as described above). Figure 1B shows that absorbance signal were obtained in | + | was employed to test the affinity of the virus particles. As displayed |
- | case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C did not | + | in |
- | yield any significant absorbance signals. In addition, the assay was performed | + | figure 1A, only Z34C-presenting particles should be immobilized and |
- | with undiluted samples and those which were 10-fold diluted in PBST + 0.5 % | + | only |
- | BSA. Signal strengths in the undiluted ELISA samples indicated that the assay | + | assembled viral capsid will be detected due to the affinity of the A20 |
- | was saturated, therefore the diluted sample data were used for evaluation. The | + | antibody |
+ | (as described above). Figure 1B shows that absorbance signal were | ||
+ | obtained in | ||
+ | case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C | ||
+ | did not | ||
+ | yield any significant absorbance signals. In addition, the assay was | ||
+ | performed | ||
+ | with undiluted samples and those which were 10-fold diluted in PBST + | ||
+ | 0.5 % | ||
+ | BSA. Signal strengths in the undiluted ELISA samples indicated that the | ||
+ | assay | ||
+ | was saturated, therefore the diluted sample data were used for | ||
+ | evaluation. The | ||
ABTS conversion indicates that highest amount of Z34C-presenting viral | ABTS conversion indicates that highest amount of Z34C-presenting viral | ||
particles is present in the Äkta fractions 7-9 of the two different 587 | particles is present in the Äkta fractions 7-9 of the two different 587 | ||
- | insertions. No viral particles with a binding affinity for the IgG antibody | + | insertions. No viral particles with a binding affinity for the IgG |
+ | antibody | ||
were present in the 453 samples.</span></p> | were present in the 453 samples.</span></p> | ||
- | + | <div align="center"> | |
- | <div align=center> | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | <table class=MsoTableGrid | + | cellpadding="0" cellspacing="0"> |
- | + | <tbody> | |
- | + | <tr style="height: 593.75pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 318.25pt; height: 593.75pt;" | |
- | + | valign="top" width="424"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img style="width: 410px; height: 793px;" alt="" | |
- | + | src="https://static.igem.org/mediawiki/2010/0/0b/Freiburg10_Z34C_titration_data.png" | |
- | + | align="left" hspace="12"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr style="height: 39.25pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 318.25pt; height: 39.25pt;" | |
- | + | valign="top" width="424"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">A: Sandwich-ELISA scheme for the | |
- | + | detection of AAV-2 particles. Immobilization is achieved by binding to | |
- | + | the A20 capture molecule which also acts as the biotinylated detection | |
- | + | antibody after washing. Intact viral capsids can be specifically | |
- | + | detected by Strepavidin-HRP</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">B: ELISA using the principle described | |
- | + | in A for loop insertion samples obtained by gel filtration | |
- | + | chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold | |
- | + | diluted samples were employed</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">C: qPCR data for the | |
- | + | CMV-promoter-based DRP-titer determination. Samples as in B</span></p> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">D: ELISA signals obtained by | |
+ | absorbance measurements from B at 405 nm, diluted samples</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | |||
</div> | </div> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US">A second approach was conducted to | |
- | <p class=MsoNormal | + | reveal the reason for the absence |
- | lang=EN-US>A second approach was conducted to reveal the reason for the absence | + | of Z34C-presenting particles in case of the 453 insertion. The sandwich |
- | of Z34C-presenting particles in case of the 453 insertion. The sandwich ELISA, | + | ELISA, |
as shown in figure 2A, uses the A20 antibody to capture and detect all | as shown in figure 2A, uses the A20 antibody to capture and detect all | ||
- | assembled viral capsids independent of the presence of a Z34C motif giving the | + | assembled viral capsids independent of the presence of a Z34C motif |
- | so-called physical AAV titer (see figure 2D). To correlate these measurements, | + | giving the |
- | we additionally conducted qPCR measurements to determine the amount of encapsidated | + | so-called physical AAV titer (see figure 2D). To correlate these |
- | vector plasmids, the so-called DRP (DNAse-resistant particle) titer (see figure | + | measurements, |
- | 2C). Comparison reveals that both assays yielded a peak signal around the Äkta | + | we additionally conducted qPCR measurements to determine the amount of |
- | fraction 8. The qPCR data indicated that all samples contain approximately 2 x | + | encapsidated |
- | 10^9 copies of the vector plasmid per milliliter. Assuming equal packaging | + | vector plasmids, the so-called DRP (DNAse-resistant particle) titer |
- | efficiencies for the vector plasmids, all three loop insertion approaches | + | (see figure |
- | contain comparable amounts of viral capsids. The sandwich-ELISA yielded a peak | + | 2C). Comparison reveals that both assays yielded a peak signal around |
- | around fraction eight for all three loop-insertion approaches. Since the signal | + | the Äkta |
- | strength for the 587KO-Z34C samples is significantly higher, it can be assumed that | + | fraction 8. The qPCR data indicated that all samples contain |
- | additionally to the affinity of the A20 antibody for assembled viral capsids, | + | approximately 2 x |
- | the Z34C-containing viral particles add a high affinity for the Fc-part of the | + | 10^9 copies of the vector plasmid per milliliter. Assuming equal |
- | detection antibody. Hence the qPCR-titration only depends on the number of | + | packaging |
- | encapsidated AAV-vector plasmids and not on the viral capsid and its degree of | + | efficiencies for the vector plasmids, all three loop insertion |
- | modification, it is the more quantitative method of particle titration which | + | approaches |
+ | contain comparable amounts of viral capsids. The sandwich-ELISA yielded | ||
+ | a peak | ||
+ | around fraction eight for all three loop-insertion approaches. Since | ||
+ | the signal | ||
+ | strength for the 587KO-Z34C samples is significantly higher, it can be | ||
+ | assumed that | ||
+ | additionally to the affinity of the A20 antibody for assembled viral | ||
+ | capsids, | ||
+ | the Z34C-containing viral particles add a high affinity for the Fc-part | ||
+ | of the | ||
+ | detection antibody. Hence the qPCR-titration only depends on the number | ||
+ | of | ||
+ | encapsidated AAV-vector plasmids and not on the viral capsid and its | ||
+ | degree of | ||
+ | modification, it is the more quantitative method of particle titration | ||
+ | which | ||
proved equal for all three loop insertion approaches.</span></p> | proved equal for all three loop insertion approaches.</span></p> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p> | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" |
- | + | align="left"><span lang="EN-US"> </span></p> | |
- | <p class=MsoNormal | + | <p class="MsoNormal" |
- | lang=EN-US> </span></p> | + | style="text-align: left; text-indent: 0cm; page-break-after: avoid;" |
- | + | align="left"><span lang="EN-US">Summarizing, the insertion of a 34/39 | |
- | <p class=MsoNormal | + | amino acid functional motif into the two most promising integration |
- | page-break-after:avoid | + | sites of |
- | amino acid functional motif into the two most promising integration sites of | + | the AAV-2 capsid (see figure 3) revealed no integration of modified |
- | the AAV-2 capsid (see figure 3) revealed no integration of modified viral | + | viral |
- | capsid proteins (VP1-3) into assembled viral capsids for the 453 integration | + | capsid proteins (VP1-3) into assembled viral capsids for the 453 |
- | site. As also seen by the data obtained for the linker-containing insertion, | + | integration |
- | the 587 region tolerates insertions of at least 39 amino acids. For the 453 | + | site. As also seen by the data obtained for the linker-containing |
- | integration site, either the inserted Z34C motif loses its ability to bind IgG | + | insertion, |
- | molecules or the influence of the inserted motif causes strong structural | + | the 587 region tolerates insertions of at least 39 amino acids. For the |
- | changes that disturb the ability of the modified VP proteins to be integrated | + | 453 |
+ | integration site, either the inserted Z34C motif loses its ability to | ||
+ | bind IgG | ||
+ | molecules or the influence of the inserted motif causes strong | ||
+ | structural | ||
+ | changes that disturb the ability of the modified VP proteins to be | ||
+ | integrated | ||
into the virus capsid.</span></p> | into the virus capsid.</span></p> | ||
- | + | <p class="MsoNormal"><span lang="EN-US"> </span></p> | |
- | <p class=MsoNormal><span lang=EN-US> </span></p> | + | <table class="MsoTableGrid" |
- | + | style="border: medium none ; border-collapse: collapse;" border="0" | |
- | <table class=MsoTableGrid | + | cellpadding="0" cellspacing="0"> |
- | + | <tbody> | |
- | + | <tr style="height: 209.35pt;"> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt; height: 209.35pt;" | |
- | + | valign="top" width="636"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><img | |
- | + | style="border: 0px solid ; width: 571px; height: 269px;" alt="" | |
- | + | id="Picture 15" | |
- | + | src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_loop_insertion_sites.png"></p> | |
- | + | </td> | |
- | + | </tr> | |
- | + | <tr> | |
- | + | <td style="padding: 0cm 5.4pt; width: 477.3pt;" valign="top" | |
- | + | width="636"> | |
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | + | align="left"><span lang="EN-US">Loop insertion sites visualized in the | |
+ | 3D structure of a single viral coat protein (VP3)</span></p> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </tbody> | ||
</table> | </table> | ||
- | + | <p class="MsoNormal" style="text-align: left; text-indent: 0cm;" | |
- | <p class=MsoNormal | + | align="left"><span lang="EN-US"> </span></p> |
- | lang=EN-US> </span></p | + | |
- | + | ||
- | + | ||
</html> | </html> | ||
{{:Team:Freiburg_Bioware/Footer}} | {{:Team:Freiburg_Bioware/Footer}} |
Revision as of 02:26, 28 October 2010
h1>His-Affinity tag</h1>
<img style="width: 395px; height: 198px;" src="" alt="" align="left" hspace="12"> |
Schematic figure of His-affinity Tag insertion into the viral capsid |
The insertion of a His-Affinity Tag into the exposed major surface loops of the viral vectors allows their specific affinity purification employing e.g. Ni-NTA affinity chromatography. This purification method was tested using the cell culture lysate of transfected AAV-293 cells that were either grown in DMEM supplemented with 10% FCS or in the serum-free GIBCO® FreeStyle™ 293 Expression Medium (Invitrogen). The usage of serum-free media is a technological modification meant to facilitate the production of pure viral vectors. Purified viral vectors are important for several applications such as animal models and biophysical characterizations. On the other hand, the elution of the His-tagged viral vectors allows also enrichment of transgene viral vectors. In order to answer the question if and to what degree viral vectors are transferred into the media, the cells were centrifugated, then divided into the pellet fraction and the supernatant. Physical cell lysis was performed for both fractions of the two produced batches (serum-free and FCS media) by performing four cycles of freeze and thaw.
<img style="width: 627px; height: 567px;" alt="" id="Bild 1" src=""> |
Experimental setup for the purification of His-tag presenting viral particles produced by AAV-293 cells in DMEM or the protein- and animal-free Freestyle medium. Purified viral particles can be detected by ELISA using an anti-poly-Histidine antibody or qPCR for encapsidated vector plasmids, respectively |
Contents
Material and Methods:
Transfection of the AAV-293 producer cells was performed in five 10 cm petri dishes with 3.6x10^6 cells, resulting in a confluency of about 70-80% according to the standard protocol either with cells grown in GIBCO® FreeStyle™ 293 Expression Medium (Invitrogen, protein- and animal-origin free) or in DMEM supplemented with 10% FCS (PAA). For transfection, the composite parts pCMV_VP123(587-His) and RepVP123(587-KO)_p5-TATA-less were used in an 1:1 ratio together with pHelper and [AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR. Cells were spun down at 200 x g for five minutes and the samples were divided into the pellet and the supernatant fractions. Physical cell lysis was performed by four cycles of freeze and thaw for all four samples. The cell lysate / supernatant fractions were incubated with 800 µl of His-Affinity Gel (kindly provided by Zymo Research, USA) at 4 °C for 18 hours with 200 rpm constant agitation. The beads were then collected in 5 ml gravity-flow columns and washed five times with one column volume of PBS each. The His-affinity gel was subsequently washed with PBS, 25 mM Imidazole to remove unspecifically bound proteins. Elution was performed in an second step with PBS, 500 mM Imidazole to elute the His-tagged viral vectors. The genomic titer of the purified viral vectors was detected via q-PCR. In an ELISA, viral vectors were captured employing the monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, DKFZ, Heidelberg) that exclusively recognizes assembled AAV capsids. His-Tags present in assembled viral capsids were subsequently detected with an HRP-tagged secondary anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). HRP presence was detected using the peroxidase substrate ABTS. Generation of blue-green color (absorption at 405 nm) was measured in a Tecan Sunrise plate reader. Sample data were blanked with the average of the non-template controls (NTC).
Results and Discussion
Presence of the His-affinity tag in the viral capsid was detected and the ELISA enabled quantification of the purification procedure efficiency. The absorbance measured for the elution fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and 0.5 for the Free Style 293-grown cells, assigning the DMEM-grown cells a five times higher production efficiency. Comparison between the cell pellet and the supernatant fractions revealed that 70 - 80% of the viral particles can be found inside the producer cells.
According to these results,
producer cells should be grown in complex media for in vitro
and cell
culture experiments. Use of serum-free produced viral vectors is
recommended
for mouse or other animal experiments and possible therapeutical
applications
where even the presence of traces amounts of fetal calf serum should be
avoided. Combination with different purification approaches such as gel
filtration chromatography using i.e. Superdex 200 columns (GE
Healthcare)
enables the production of highly purified viral vectors for several
different
applications.
<img style="width: 451px; height: 796px;" alt="" src="" align="left" hspace="12"> |
A: Schematic overview of the sandwich ELISA for the detection of His-tagged viral particles B: ELISA from viral particles produced by AAV-293 cells in DMEM or Free Style medium, divided into cell pellet and cell culture supernatant samples. The particles were purified using Ni-NTA affinity chromatography with Imidazole in PBS as washing and elution agent C: Absorption measurements from plate shown in B. Undiluted Äkta fractions converted ABTS peroxidase substrate at 405 nm D: As C, whereas Äkta fractions were 10-fold diluted |
Biotinylation Acceptor
Peptide (BAP)
<img style="width: 419px; height: 244px;" src="" alt="http://partsregistry.org/wiki/images/a/a9/Freiburg10_ViralBrick_motif_BAP.png" align="left" hspace="12"> |
Schematic overview of BAP insertion into AAV-2 viral capsids, followed by biotinylation and binding of streptavidin-coupled molecules |
The BAP (Biotinylation Acceptor Peptide) included in the Virus Construction Kit is a 15 amino acid peptide identified by Schatz et al. (1993) in a library screening approach and published under the number #85. This peptide with the sequence GLNDIFEAQKIEWHE contains a central lysine residue that can be specifically biotinylated by the prokaryotic holoenzyme biotin synthetase, encoded by the BirA gene of E. coli. Specific biotinylation of this peptide sequence can be performed in vivo by cotransfecting a plasmid with the BirA gene as described for the AAV by Arnold et al. (2006) or by an in vitro coupling approach using the purified Escherichia coli enzyme biotin ligase (BirA).
Biotin molecules specifically coupled to the viral loops can either be used to attach Streptavidin-coupled molecules to the viral capsid or to chemically couple molecules with a reactive group to biotin. Consequently, the inserted motif can enable the visualization of single virus particles by coupling fluorophores to the virus capsid empowering further uses of the Virus Construction Kit in fundamental virological research. In addition, targeting molecules such as Streptavidin-coupled affinity molecules (i.e. Antibodies, Nanobodies or Affibodies) can be coupled for manifold targeting approaches.
Materials
and Methods:
Transfection of the AAV-293 producer cells was performed in five 10 cm petri dishes with 3.4x10^6 cells (grown in DMEM supplemented with 10% FCS) resulting in a confluence of 70-80% according to the standard protocol. Producer cells were harvested and together with the culture supernatant subjected to four cycles of freeze and thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 minutes and concentrated in a VivaSpin VS2002 column with 10 kDa molecular weight cut-off to yield two milliliters. Genomic DNA attached to the virus particles was degraded using 250 units of Benzonase (EC 3.1.30.2, Sigma-Aldrich) at 37 °C for one hour. The concentrated cell lysate was washed three times with 4 ml 20 mM bis-Tris buffer pH 6.0, 100 mM NaCl. From this concentrated sample, 500 µl were loaded on the ÄKTA purifier (GE Healthcare) equipped with a Superdex 200 gel filtration column (GE Healthcare). This purification is also described by Smith et al. (2003). Fractions around the void volume giving a UV absorbance peak were pooled and applied to a Amicon Ultra column (Millipore, size limit 100 kDa, 2 ml loading volume) and concentrated to 500 µl. This purified virus sample was washed four times with 10 mM Tris-buffer pH 8.0 buffer which is recommended for the BirA biotin ligase that was kindly provided by Avidity LCC (Colorado, USA).
The BAP-containing viral vector sample (500 µl) was mixed according to the manufacturer protocol with each 72 µl Biomix A, Biomix B and Biotin. To reach maximal biotinylation, a volume of 5 µl containing 25000 units of the biotin ligase BirA was added to the reaction mixture and incubated for 6 h at 30 °C. In order to remove unbound biotin, the biotinylated viral vectors were washed five times with 10 mM Tris buffer pH 8.0.
Biotinylation was verified with an ELISA as depicted in figure A. For the detection of successfully biotinylated viral vectors, MaxiSorp 96-well plates (Nunc) were coated with 200 ng monoclonal A20 antibody per well for eight hours at 4°C, and blocked over night with PBST + 0.5 % BSA. This plate was incubated for 1 h at room temperature with 100 µl of serial diluted viral vectors. After washing the plates three times, the recommended amount of Streptavidin-HRP conjugate (Sigma-Aldrich) was added to each well for 1 h at room temperature. Again, the plates were washed three times followed by detection of absorbance caused by converted ABTS substrate at 405 nm. Additionally, the genomic AAV-2 titer was determined by qPCR.
<img style="width: 587px; height: 539px;" alt="" src="" align="left" hspace="12"> |
A: Schematic overview of the detection of BAP-presenting viral particles immobilized by A20 capture antibodies. Biotinylated capsids can be detected by streptavidin-coupled HRP B: ELISA AAV-2 particles carrying a BAP insertion in loop 587 which can be specifically biotinylated in vitro |
Results
and Discussion:
As can be seen in figure B, biotinylation of assembled AAV particles was achieved with the dilution series ranging from 2- to 128-fold. Correlating this assay with the qPCR experiment for the detection of encapsidated vector plasmid (genomic titer using CMV-promoter primers) yields a value of 4.70E+07 DNase-resistant particles (DRP). Consequently, the presence of approximately 3.6x10^5 biotinylated DRPs can be detected using the described ELISA. Compared to the virus particle detection ELISA emploing A20 as capture- and detection antibody, the detection limit of the biotinylated viral capsids is about 10fold more sensitive. This may be explained by the higher affinity of strepavidin towards biotin relative to the A20 antibody affinity or multiple biotinylation of a single virus particle.
Miniaturized antibody
binding domain (Z34C)
<img style="width: 451px; height: 225px;" src="" alt="" align="left" hspace="12"> |
Schematic overview of Z34C insertion into AAV-2 viral capsids enabling antibody arming for therapy |
The idea of this targeting approach is to utilize a minimized fragment of the <a href="http://www.ncbi.nlm.nih.gov/protein/153106">Staphylococcal Protein A</a> that was first described in Staphylococcus aureus. These gram-positive bacteria have evolved the 508 amino acid long protein A that has a high affinity for the Fc-domain of antibodies to protect itself from the immune system. Binding to the constant region of the antibodies is accomplished by the <a href="http://www.ncbi.nlm.nih.gov/protein/1Q2NA">Z-Domain</a> of Protein A that is 58-59 amino acids long, has alone a high affinity (Kd= 14,9 nM) for the antibodies and a three-helix bundle structure. In <a href="http://www.ncbi.nlm.nih.gov/pubmed/8650153">[Braisted & Wells; 1996]</a> the authors reduced the secundary structure to an two-helix bundle. This size reduction has lead to an drastic reduction of the affinity for IgG (>10^5 fold) which could be recovered by 13 amino acid exchanges resulting in a 38 amino acid long peptide with an satisfying affinity for IgG (Kd = 185 nM) termed <a href="http://www.ncbi.nlm.nih.gov/protein/1ZDAA">Z38</a>. This binding domain was subsequently improved in <a href="http://www.ncbi.nlm.nih.gov/pubmed/9294166">[Starovasnik et al.; 1997]</a> by the insertion of a disulfide bridge connecting the ends of the helices leading to the binding domain <a href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA">Z34C</a> which shows an increased affinity for IgG (Kd = 20 nM).
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Development of the Z34C motif, a miniaturized antibody binding motif |
This engineered antibody binding domain of 34
amino
acids was then inserted into capsids of different viral vectors amongst
others
also the AAV. In <a
href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC155067">[Ried
et al.; 2002]</a> the <a
href="http://www.ncbi.nlm.nih.gov/protein/1ZDCA">Z34C</a> domain
was inserted at position 587 into the capsid of the AAV resulting in
viral
vector that can be targeted to different target cells without genetic
engineering. This targeting approach was then improved in <a
href="http://www.ncbi.nlm.nih.gov/pubmed/15922956">[Gigout
et al.; 2005]</a> by the creation of
mosaic vectors that contain only ~25% of
recombinant VP-Proteins what resulted in 4 to 5 orders of magnitude
more
infectiosity compared to all-mutant viruses.
Material and Methods:
Transfection of the AAV-293 producer cells was performed for three different loop insertions of Z34C in each three 10 cm petri dishes with 3.4x10^6 cells resulting in a confluence of about 70-80%. For the transfection, either the composite parts pCMV_VP123(453-Z34C), pCMV_VP123(587KO- Z34C) or pCMV_VP123(587Ko- Z34C-Spacer) were cotransfected with pHelper, [AAV2]-left-ITR_pCMV_betaglobin_mVenus_hGH_[AAV2]-right-ITR and RepVP123(587KO). The producer cells were harvested with the culture supernatant and subjected to four cycles of freeze and thaw cell lysis. The cell lysate was centrifuged at 4000 rpm for 15 minutes and concentrated in a VivaSpin VS2002 (Sartorius Stedem) with a 10 kDa molecular weight cut-off to two milliliters and washed three to five times with 20 mM Bis-Tris buffer, pH 6. The cell lysates were incubated with 250 units of Benzonase (Sigma-Aldrich) to remove contaminant genomic DNA and loaded on an ÄKTA purifier (GE Healthcare) equipped with a Superdex 200 gel filtration column (GE Healthcare). Fractions (500 µl) around the void volume containing viral particles were collected and used in a sandwich ELISA. 96 well plates were coated with 200 ng Cetuximab (Imclone/Merck/Bristol-Myers Squibb). Detection was performed using the monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, DKFZ Heidelberg) that was biotinylated using a Biotinylation kit (Dojindo, Japan) and Streptavidin-HRP (Sigma-Aldrich). The HRP presence was detected by the conversion of the substrate ABTS at 405 nm. The average of the non-template controls (NTC) was subtracted from the sample data.
Results and Discussion:
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A: Sandwich-ELISA scheme for the detection of Z34C-presenting viral particles. Immobilization is achieved by binding a IgG molecule. Intact viral capsids can be specifically bound by the biotinylated A20 antibody which can be detected by Strepavidin-HRP B: ELISA using the principle described in A for loop insertion samples obtained by gel filtration chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold diluted samples were employed C: ELISA signals obtained by absorbance measurements from B at 405 nm, undiluted samples D: ELISA signals obtained by absorbance measurements from B at 405 nm, diluted samples |
Three samples of different viral particles with insertions of the antibody-binding motif Z34C were successfully purified by the gel filtration chromatography. The fractions around the void volume were subsequently used in two different sandwich ELISAs. The first one aims at the detection of the particle’s ability to bind IgG-antibodies. The therapeutical antibody Cetuximab was employed to test the affinity of the virus particles. As displayed in figure 1A, only Z34C-presenting particles should be immobilized and only assembled viral capsid will be detected due to the affinity of the A20 antibody (as described above). Figure 1B shows that absorbance signal were obtained in case of the 587KO-Z34C and 587KO-Z34C-spacer insertions. Loop 453-Z34C did not yield any significant absorbance signals. In addition, the assay was performed with undiluted samples and those which were 10-fold diluted in PBST + 0.5 % BSA. Signal strengths in the undiluted ELISA samples indicated that the assay was saturated, therefore the diluted sample data were used for evaluation. The ABTS conversion indicates that highest amount of Z34C-presenting viral particles is present in the Äkta fractions 7-9 of the two different 587 insertions. No viral particles with a binding affinity for the IgG antibody were present in the 453 samples.
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A: Sandwich-ELISA scheme for the detection of AAV-2 particles. Immobilization is achieved by binding to the A20 capture molecule which also acts as the biotinylated detection antibody after washing. Intact viral capsids can be specifically detected by Strepavidin-HRP B: ELISA using the principle described in A for loop insertion samples obtained by gel filtration chromatography of 453- and 587-Z34C particles. Undiluted and 10-fold diluted samples were employed C: qPCR data for the CMV-promoter-based DRP-titer determination. Samples as in B D: ELISA signals obtained by absorbance measurements from B at 405 nm, diluted samples |
A second approach was conducted to reveal the reason for the absence of Z34C-presenting particles in case of the 453 insertion. The sandwich ELISA, as shown in figure 2A, uses the A20 antibody to capture and detect all assembled viral capsids independent of the presence of a Z34C motif giving the so-called physical AAV titer (see figure 2D). To correlate these measurements, we additionally conducted qPCR measurements to determine the amount of encapsidated vector plasmids, the so-called DRP (DNAse-resistant particle) titer (see figure 2C). Comparison reveals that both assays yielded a peak signal around the Äkta fraction 8. The qPCR data indicated that all samples contain approximately 2 x 10^9 copies of the vector plasmid per milliliter. Assuming equal packaging efficiencies for the vector plasmids, all three loop insertion approaches contain comparable amounts of viral capsids. The sandwich-ELISA yielded a peak around fraction eight for all three loop-insertion approaches. Since the signal strength for the 587KO-Z34C samples is significantly higher, it can be assumed that additionally to the affinity of the A20 antibody for assembled viral capsids, the Z34C-containing viral particles add a high affinity for the Fc-part of the detection antibody. Hence the qPCR-titration only depends on the number of encapsidated AAV-vector plasmids and not on the viral capsid and its degree of modification, it is the more quantitative method of particle titration which proved equal for all three loop insertion approaches.
Summarizing, the insertion of a 34/39 amino acid functional motif into the two most promising integration sites of the AAV-2 capsid (see figure 3) revealed no integration of modified viral capsid proteins (VP1-3) into assembled viral capsids for the 453 integration site. As also seen by the data obtained for the linker-containing insertion, the 587 region tolerates insertions of at least 39 amino acids. For the 453 integration site, either the inserted Z34C motif loses its ability to bind IgG molecules or the influence of the inserted motif causes strong structural changes that disturb the ability of the modified VP proteins to be integrated into the virus capsid.
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Loop insertion sites visualized in the 3D structure of a single viral coat protein (VP3) |
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