Team:METU Turkey/Results Discussion/CooA Expression and Purification

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<p style="font-size:110%; color:#576f91; font-family:georgia,serif;"><br>  
<p style="font-size:110%; color:#576f91; font-family:georgia,serif;"><br>  
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<br> <h2>1-Overexpression optimization</h2>
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<br>1-Overexpression optimization
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<br> <h3>A. Expression vector comparison (pKK versus pTriEx)</h3>
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<br>When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector
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<br>A. Expression vector comparison (pKK versus pTriEx)
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<br> <h3>A.1. Expression in BL21 with pTriEx</h3>
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<br> In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
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<br>When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector.  
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/3/35/Br1.jpg"></a></div>
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<br>10/09/18
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<br> <h3>A.2. Expression in JM109 and BL21 with pKK vector</h3>
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<br>Expression in BL21 with pTriEx
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We failed to express pKK vector in JM109. We have a little expression in BL21.
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<br>In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/b9/Br2.jpg"></a></div>
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<br>10/10/05
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/pKK_versus_pTriEx"><strong>Details...</strong></a></div></html>
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<br>Expression in JM109 and BL21 with pKK vector
 
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<br>In this expriment, for the first time we want to try CooA pKK vector in BL21. The vector was sent by Aono and he advised us to express it in JM109. But we have failed to express it. So we tried it in BL21. We have no result with JM109 and a little expression in BL21. Therefore; we gave up from expressing pKK vector in BL21. And JM109 grows slowly,also we never see any band in JM109 crude lysate in many unpublished data. Therefore; we lysated all bacteria with same OD600. But result wasn't changed.
 
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<br> <h3>B. Expression host comparison (JM109 versus BL21)</h3>
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<br>B. Expression host comparison (JM109 versus BL21)
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<br> BL21 is better host than JM109 for CooA.
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<br>When we look at the experiment 10/10/5, there is no expression with pKK in JM109 and we have greyed bands belongs to CooA as indicated gel photo of 10/10/5 experiment.
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<br> <h3>C. IPTG induction studies</h3>
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<br> Expression comparison with different IPTG Concentration of pTriEx vector.
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/6/6a/Br3.jpg"></a></div></html>
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<br> Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.
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<br>C. IPTG induction studies
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/IPTG_induction"><strong>Details...</strong></a></div></html>
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<br>Expression comparison with different IPTG Concentration of pTriEx vector
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<br> <h3>D. Ferric Citrate effect</h3>
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<br>10/10/10
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<br> Time Dependent Expression of pTriEx vector  with Ferric Citrate and without Ferric Citrate
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<br>In expression comparison with IPTG concentration experiment, we realized that our CooA pTriEx vector is expressed without IPTG induction however when the cultures were induced with IPTG the expression incresed a little. Thus, we concluded that IPTG concentration doesn't much affect our expression yield. However, when 2mM IPTG was added to culture,expression of CooA was decreased. It has probably a toxic affect on our bacteria.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/5/5f/Br4.jpg"></a></div></html>
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<br>D. Ferric Citrate effect
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<br> According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.
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<br>Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Ferric_Citrate_effect"><strong>Details...</strong></a></div></html>
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<br>10/10/10
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<br> <h3>E. Soluble versus insoluble fraction</h3>
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<br>In the comparison of the cultures with Ferric Citrate and without Ferric Citrate, we prepared different cultures that all of them induced with IPTG and 4 of them had Ferric Citaret adn 4 of them did not have Ferric Citrate. According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. In this expreiment we also determine the best IPTG induction time. The cultures were induced for 3-5-7-9 hours. At the end of the experiment we concluded that the induction time also affects expression of CooA, however 5 hour induction is the best because both monomer and dimer form of CooA is highly expressed and the impurities are less when compared to 9 hours induction. In -FC7 well, we probably made pipeting error; therefore, we had greyed bands.
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<br> Lysis Buffer with Urea and without Urea
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<br>E. Soluble versus insoluble fraction
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/4/43/Br5.jpg"></a></div></html>
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<br>Lysis Buffer with Urea and without Urea
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<br> Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.
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<br>10/09/23
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/Soluble_vs_insoluble_fraction"><strong>Details...</strong></a></div></html>
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<br>BL21 with pKK vector
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<br> <h2>2-Purification optimization</h2>
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<br>JM109 with pKK vector
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<br>Objectives:
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<br> <h3>A. Ion exchange chromatography with Sepharose Q (QS)</h3>
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<br>In this experiment, we wanted to understand whether the CooA accumulates in inclusion body. In order to understand that we added urea in our lysis buffer.
+
 +
<br> Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.
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<br>We subcultured our cells over night and then passed to scale up. After cultures reached to 0.5-0.6 value at OD600 then the cultures were induced with 1 mM IPTG for 5 hours. After induction, we centrifuged the cultures at 6000g for 15 minutes and lysate our pellets with lysis buffer. Lysis buffer includes 50 mM Tris-HCl- 5 mM EDTA, 100 mM NaCl, 8M Urea, 1.7 mM dithionite, 1 mM DTT, 7.5 uL protease inhibitor. The ph of buffer is important so we adjusted the lysis buffer to pH 7.5. The resuspended cells were lysated with probe sonicator. After the sonication process, the cultures were centrifuged at 20000g for 20 minutes. And, the samples were loaded to SDS-Page. We compared results of lysated cultures with lysis buffer that contains urea and with lysis buffer that does not contain urea.
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<br> <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/QS_results"><strong>Details...</strong></a></div></html>
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<br>Results:
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<html>
-
<br>As seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/1/14/Br13.jpg"></a></div></html>
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<br>2.Purification optimization
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/7/76/B30.jpg"></a></div></html>
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<br> A. Ion exchange chromatography with Sepharose Q (QS)
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<html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/9/93/Br14.jpg"></a></div></html>
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<br>10/09/26
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<br> <h3>B. Affinity chromatography with chealating sepharose(CS)</h3>
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<br>Purification with QS BL21 pTriEx
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<br> Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.
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<br>This was the first experiment of BL21 pTriEx with FPLC. We obeyed the general protein purification procedure except sonication. We lysed pellets with French Press. Then we centrifued the crude lysate at 32000 g for 60 min. We took a sample for SDS-PAGE. Then we continue with filtration. Filtered crude lysate was looaded to Q-sepharose column. According to FPLC graph, we have a sharp peak at 280nm which represents proteins. We picked up the tubes between 9 and 14. SDS-Page shows the sharp peak that contains CooA. F11 is the top of the sharp peak. We also check small peak which corresponds to 46th tube. We combined the F10, F11 and F12 elutes for further purification with chelating sepharose column.
 
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<br>  <html><div><a href="https://2010.igem.org/Team:METU_Turkey/Results_Discussion/CS"><strong>Details...</strong></a></div></html>
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/7/74/Br15.jpg"></a></div></html>
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<br> <h2>C. Concentrated CooA</h2>
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<br> CooA protein contains heme group therefore its solution is seen yellow to orange
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<div align="center" style="margin:15px 0px 0px 0px"><img style="border: 0px solid ; width: 400px; height: 400px;" alt="w7" src="https://static.igem.org/mediawiki/2010/b/be/Br18.jpg"></a></div></html>
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Latest revision as of 03:39, 28 October 2010

Home

 


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3-CooA Expression and Purification



1-Overexpression optimization


A. Expression vector comparison (pKK versus pTriEx)


When we compare the below results, the expression of pTriEx vector is higher than the expression of pTriEx vector

A.1. Expression in BL21 with pTriEx


In this expression experiment, we obtained 2 different bands in BL21 pTriEx from BL21 negative control as indicated gel photo. CooA is found in two form (monomer and dimer).
w7

A.2. Expression in JM109 and BL21 with pKK vector

We failed to express pKK vector in JM109. We have a little expression in BL21.
w7



Contents

B. Expression host comparison (JM109 versus BL21)


BL21 is better host than JM109 for CooA.


C. IPTG induction studies


Expression comparison with different IPTG Concentration of pTriEx vector.

w7


Our CooA pTriEx vector is expressed without IPTG induction but is less than others. When 2mM IPTG was added to culture, expression of CooA was decreased. It has probably a toxic effect on our bacteria.



D. Ferric Citrate effect


Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate

w7


According to the SDS-Page result, Ferric Citrate increses the expression of CooA. It is probably releated to heme characteristic of CooA. The best IPTG induction time among 3-5-7-9 hours is 5 hour.



E. Soluble versus insoluble fraction


Lysis Buffer with Urea and without Urea

w7


Results are seen in the SDS-Page photograph, there is no difference between bands of same samples (look at all the 50kDa bands). Thus,we concluded that CooA does not accumulate in inclusion body.



2-Purification optimization


A. Ion exchange chromatography with Sepharose Q (QS)


Purification QS with BL21 pTriEx: Yellowish color indicates our CooA protein. Then we looaded 27,28,29,30 and 31 to SDS-Gel the elutes which have a high value at 420nm because oxidized CooA gives a Sorret peak at 420 nm according to Aono. CooA concentration was determined according to extinction coefficient of heme (Ferric State) which is 108 mM-1 cm-1 according to Aono. The SDS gel indicates that our protein is colose to 50 kDa in dimer. MW of monomer is estimated 24-25kDa according to Aono.



w7

w7

w7


B. Affinity chromatography with chealating sepharose(CS)


Purification with CS BL21 pTriEx: Loaded combined FPLC tubes from QS column(10/09/26) gave two close picks in CS column. First one corresponds to the CooA. The elutions of CooA are C13,C14 and C15. We combined them for further analyzes with EMSA and ITC and stored @+4. C16 AND C17 are combined to reload to CS column because they are contaminated another protein which is close to 40kDa.



w7

w7



C. Concentrated CooA


CooA protein contains heme group therefore its solution is seen yellow to orange

w7




-- </br>

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