大肠杆菌二硫键异构酶DsbC的表达、纯化及活性分析  

Characterization of Escherichia coli Disulfide Isomerase DsbC

作　　者：许成钢[1] 付月君[1] 梁爱华[1] 

机构地区：[1]山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,山西太原030006

出　　处：《山西大学学报（自然科学版）》2008年第2期258-261,共4页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(30700534)

摘　　要：将大肠杆菌二硫键异构酶DsbC基因克隆到表达载体pRSETc中,并在宿主菌E.coli BL21(DE3)中通过IPTG诱导进行可溶性表达.用Ni-NTA亲和层析柱从菌体裂解液中纯化了His6-DsbC,通过Superdex 75凝胶过滤层析获得纯His6-DsbC蛋白.Western blot和凝胶过滤层析分析表明,该蛋白以二聚体的形式存在.通过RP-HPLC分析,表达的His6-DsbC以氧化型和还原型存在,并且该两种形态在一定的氧化还原体系中能够相互转化.HRP重折叠实验说明,重组DsbC能够提高HRP的复性率.A disulfide bonds isomerase gene from Escherichia coli, DsbC, was cloned into the expression vector pRSETc, and expressed in E. coli BL21 （DE3） host cells. The fusion protein, His6-DsbC, was expressed in soluble form and was purified to give a single band on SDS-PAGE by affinity chromatography. The Western blot analysis and gel filtration chromatography demonstrated that this protein was rDsbC, and it was in a dimer form. The recombinant DsbC was proved to have redox activity and refolding ability by analysis of reversed-phase HPLC and refolding assay of HRP, respectively.

关 键 词：ESCHERICHIA coli二硫键异构酶DsbC 原核表达 二聚体 氧化还原活性 辣根过氧化物酶(HRP)重折叠 

分 类 号：Q814[生物学—生物工程]