文献类型：期刊文献

中文题名：星海链霉菌卤化酶重组蛋白在大肠杆菌中可溶表达

英文题名：Soluble expression of halogenase SinH fromStreptomyces xinghaiensis in Escherichia coli

作者：王玉梅[1];赵心清[1];马昱澍[2];魏东芝[2]

机构：[1]大连理工大学生命科学与技术学院,辽宁大连116024;[2]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2014

卷号：54

期号：3

起止页码：278

中文期刊名：大连理工大学学报

外文期刊名：Journal of Dalian University of Technology

收录：CSTPCD;;Scopus;北大核心:【北大核心2011】；CSCD:【CSCD2013_2014】；

基金：华东理工大学生物反应器工程国家重点实验室开放课题资助项目;中国科学院热带海洋生物资源与生态重点实验室开放课题资助项目(LMB111002)

语种：中文

中文关键词：卤化酶;星海链霉菌;可溶表达;泛素标签;链霉菌分子伴侣;共表达体系;蛋白纯化

外文关键词：halogenase;Streptomyces xinghaiensis;soluble expression;ubiquitin tag;Streptomyces chaperonin;coexpression system;protein purification

摘要：大肠杆菌(Escherichia coli,E.coli)是表达异源蛋白的良好宿主,但常遇到蛋白表达产物不可溶的困难.在对来自星海链霉菌的卤化酶基因sinH进行大肠杆菌异源表达时,为克服蛋白表达产物可溶性低的问题,探讨了不同载体对蛋白可溶表达的影响.利用pET28a进行SinH表达时不能得到可溶的目标蛋白;使用含有泛素标签及内含肽的载体pHUIE实现了卤化酶蛋白SinH的可溶表达,但纯化后纯度不高;利用带有链霉菌分子伴侣蛋白基因的载体pET28a-SinH与pETcoco-pL1SL2在E.coli BL21(DE3)中共表达,在30℃,0.1mmol/L IPTG诱导条件下表达4h,成功获得大量可溶蛋白,使用亲和层析(Ni-NTA)纯化,获得了较纯的目标蛋白SinH,上述研究结果为在大肠杆菌中进行其他链霉菌蛋白的可溶表达提供了参考.
Escherichia coli（E.coli）is a good host for expression of heterologous proteins,but insoluble protein products are often obtained.In order to overcome the problem of low soluble expression,halogenase gene sinHfromStreptomyces xinghaiensis is expressed by E.coli,the effect of different vectors on the solubility is discussed.No soluble target protein is detected when it is expressed using pET28a,whereas by using expression vector pHUIE containing ubiquitin tag and intein,soluble target protein is obtained and purified,but the purity is low.The coexpression system containing pET28a-SinH and pETcoco-pL1SL2, which carries genes encoding Streptomyces chaperonin,is constructed in E.coli BL21（DE3）,and the optimized induction condition is established as 0.1mmol/L IPTG for induction,and expression time is 4hunder 30 ℃.The target protein is purified by affinity chromatography Ni-NTA,and pure SinH protein is obtained.The above research results provide basis for further exploration of soluble protein expression of Streptomyces in E.coli.