重组融合蛋白MBP-PAI的表达、纯化及酶活测定

为了解决亚油酸异构酶(Linoleic Acid Isomerase,PAI)在大肠杆菌中形成包涵体的问题,选择麦芽糖结合蛋白(Maltose-Binding Protein,MBP)标签和低温诱导表达系统pColdV,构建了大肠杆菌重组表达菌株E.coli BL21(pCold-Mpai),并对其诱导表达条件进行了优化。SDS-PAGE电泳结果显示,融合蛋白MBP-PAI成功表达,重组菌的最佳诱导表达条件为:诱导温度15℃、IPTG添加量0.1 mmol/L、诱导时间12 h,在该诱导条件下,与未融合MBP的PAI相比,MBP-PAI的可溶性表达量为后者的18倍、酶活为后者的1.5倍。经过MBPTrap HP亲和层析柱纯化后,MBP-PAI纯蛋白质的比酶活为1.58 U/mg,能够转化亚油酸形成反10,顺12-共轭亚油酸。

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

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.