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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
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).
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.
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.
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 affect on our bacteria.
D. Ferric Citrate effect
Time Dependent Expression of pTriEx vector with Ferric Citrate and without Ferric Citrate
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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
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.
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.
C. Concentrated CooA
CooA protein contains heme group therefore its solution is seen yellow to orange
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