hTNFR1在大肠杆菌中可溶性表达及纯化  

Soluble Expression and Purification of hTNFR1 in Escherichia coli

作　　者：韩平 盖园明 朱蓓薇[1] 张大伟[2] HAN Ping;GAI Yuanming;ZHU Beiwei;ZHANG Dawei(School of Food Science and Technology, Dalian Polytechnic University, Liaoning Dalian 116034, China;Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China)

机构地区：[1]大连工业大学食品学院,辽宁大连116034 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《生物技术进展》2018年第3期246-253,共8页Current Biotechnology

基　　金：天津市杰出青年科学基金(17JCJQJC45300);天津市自然科学基金(16JCYBJC23500);中国科学院系统微生物工程重点实验室开放课题资助

摘　　要：将人源肿瘤坏死因子Ⅰ型受体(hTNFR1)基因克隆到p ET-22b表达载体,成功构建了重组表达质粒pETH1,电转到Escherichia coli BL21(DE3)表达菌株中进行摇瓶发酵。实现了hTNFR1在大肠杆菌表达系统中的重组表达。但目的蛋白全部以包涵体的形式存在于沉淀中。为了提高hTNFR1在大肠杆菌中的可溶性表达,融合标签和分子伴侣两种策略被实施用于辅助hTNFR1的可溶性表达。结果表明,在hTNFR1的N端融合Nus A标签后,hTNFR1的可溶性有一定提高;在Nus A-hTNFR1基础上,过表达了7种分子伴侣,筛选出tig分子伴侣对hTNFR1蛋白可溶性表达有明显的促进作用,可溶性表达量约占总量的90%;对优化后的hTNFR1表达系统的可溶性蛋白进行Ni-NTA亲和层析纯化后,TEV蛋白酶酶切去除N端的Nus A标签,结合Western blot分析鉴定,获得了大量高纯度的hTNFR1蛋白。研究结果为进一步研究hTNFR1的生理学活性及其在疾病治疗方面的应用奠定了良好基础。The human tumor necrosis factor type 1 receptor（ hTNFR1） gene was cloned into pET-22 b expression vector,this study successfully constructed the recombinant plasmid p ETH1 and transformed it into Escherichia coli BL21（ DE3） expression strain for shake flask fermentation,realizing heterologous expression of hTNFR1 in E. coli. However,all the proteins were in the forms of inclusion bodies. To improve the soluble expression of the target protein existed in the sedimentation in E. coli,we used two strategies of fusion tagging and molecular chaperone for protein folding. Results showed that,the fusion of Nus A to the Nterminus of hTNFR1 increased the solubility; based on Nus A-hTNFR1,seven molecular chaperones was overexpressed,among which the molecular chaperone tig was screened for it showed significant promotion on the soluble expression of hTNFR1 in E. coli; we purified protein by Ni-NTA affinity chromatography,then,the tig of Nus A in the N-terminus of hTNFR1 was removed by enzymatic hydrolysis with TEV protease. After identification by Western blot,the hTNFR1 protein with high purity was obtained.The results laid a good foundation for further study on the physiological activity of hTNFR1 as well as the treatment of diseases.

关 键 词：hTNFR1 大肠杆菌 NusA标签 tig分子伴侣 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]