重组融合蛋白MBP-PAI的表达、纯化及酶活测定

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

DOI：10.3969/j.issn.1673-1689.2020.02.002

中文关键词: 亚油酸异构酶 大肠杆菌 包涵体 麦芽糖结合蛋白 蛋白质纯化

英文关键词: linoleic acid isomerase,Escherichia coli,inclusion body,maltose binding protein,protein purification

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中文摘要:

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.coliBL21(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。

英文摘要:

In order to reduce the inclusion bodies of the linoleic acid isomerase（PAI）in Escherichia coli，a recombinant expression strainE.coliBL21（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.

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