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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.
10/09/18
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).
10/10/05
Expression in JM109 and BL21 with pKK vector
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 And JM109 grows slowly,also we never see any band in JM109 crude lysate in many unpublished data.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.
B. Expression host comparison (JM109 versus BL21)
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.
C. IPTG induction studies
Expression comparison with different IPTG Concentration of pTriEx vector
10/10/10
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.
D. Ferric Citrate effect
Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate
10/10/10
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.
E. Soluble versus insoluble fraction
Lysis Buffer with Urea and without Urea
10/09/23
BL21 with pKK vector
JM109 with pKK vector
Objectives:
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.
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.
Results:
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.
2.Purification optimization
A. Ion exchange chromatography with Sepharose Q (QS)
10/09/26
Purification with QS BL21 pTriEx
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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