鳉鱼肠激酶在大肠埃希菌中的可溶性表达、纯化及活性鉴定  

Soluble expression of medaka enterokinase in E.coli and purification and activity of expressed product

作　　者：张五妮 刘玉林 朱秋媚 王江林 毕江坤 俞露 张宇 ZHANG Wu-ni;LIU Yu-lin;ZHU Qiu-mei;WANG Jiang-lin;BI Jiang-kun;YU Lu;ZHANG Yu(Department of Cytokines,Changchun Institute of Biological Products Co.,Ltd.,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所有限责任公司细胞因子室,吉林长春130012

出　　处：《中国生物制品学杂志》2022年第6期678-684,共7页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(20160204034YY)。

摘　　要：目的利用大肠埃希菌表达系统实现鳉鱼肠激酶(enterokinase,EK)的可溶性表达,经纯化后获得具有酶切活性的重组鳉鱼EK。方法通过基因工程技术,构建pGEX-EK重组质粒,转化至TransB(DE3)化学感受态细胞,IPTG诱导表达获得可溶的GST-EK融合蛋白,经谷胱甘肽(glutathione,GSH)亲和层析、离子交换层析,获得EK,并进行活性分析及酶切特异性试验。结果pGEX-EK重组质粒经双酶切及测序鉴定证明构建正确,经可溶性标签实现了具有生物活性的鳉鱼EK的可溶性表达,酶比活性为46 IU/mg,且具有较高的酶切特异性。结论通过使用GST融合标签成功实现了鳉鱼EK在大肠埃希菌中的可溶性表达,为实现EK的可溶性表达提供了新思路。Objective To express medaka enterokinase(EK)in a soluble form in E.coli and purify the expressed product to obtain the recombinant medaka EK with enzyme digestion activity.Methods Recombinant plasmid pGEX-EK was constructed by gene engineering technology and transformed to competent E.coli TransB(DE3)cells and induced with IPTG.The expressed soluble GST-EK fusion protein was purified by glutathione(GSH)affinity chromatography and ion exchange chromatography,and the obtained EK was analyzed for enzyme digestion activity and specificity.Results Both restriction analysis and sequencing proved that recombinant plasmid pGEX-EK was constructed correctly.Medaka EK was expressed in a soluble form by using a soluble tag,which showed a specific activity of 46 IU/mg and a high specificity.Conclusion Medaka EK was successfully expressed in a soluble form in E.coli by using GST fusion tag,which provided a route for soluble expression of EK.

关 键 词：肠激酶 鳉鱼 大肠埃希菌 可溶性表达 融合标签 

分 类 号：Q812[生物学—生物工程]