Expression, Purification and Enzyme Activity Determination of Recombination Fusion Protein MBP-PAI

Abstract:

In order to reduce the inclusion bodies of the linoleic acid isomerase（PAI）in Escherichia coli，a recombinant expression strain E.coli BL21（pCold-Mpai）was constructed by using maltose-binding protein（MBP）and low temperature induced expression system pColdV，and its induction conditions were optimized. SDS-PAGE showed that the fusion protein MBP-PAI was successfully expressed，and the best induction conditions were: induction temperature 15 ℃，IPTG 0.1 mmol/L and induction time 12 h. Under the best induction conditions，the soluble expression and enzyme activity of MBP-PAI was 18 times and 1.5 times that of PAI，respectively. After purification by MBPTrap HP affinity chromatography column，the enzyme activity of MBP-PAI was 1.58 U/mg，which could convert linoleic acid to trans10，cis12-conjugated linoleic acid.