大肠杆菌分子伴侣表达的辅助载体构建及应

摘要

异源蛋白在大肠杆菌不能正确折叠表达成包涵体,分子伴侣能在细胞内帮助异源蛋白折叠,改善蛋白的聚集.本研究以大肠杆菌(Escherichia coli) DH5α菌株DNA为模板,利用PCR技术扩增出6个分子伴侣编码基因groEL、groES、dnaK、dnaJ、grpE和clpB,分别将groEL,groES和grpE( I组)插入pCDFDuet-1载体,将dnaK、dnaJ和c1pB( II组)基因插入pRSFDuet-1,构建辅助载体pR-GESP和pC-DJKL,每个重组载体中含有一个人工操纵子,每个基因上游含有T7启动子.SDS-PAGE结果显示,诱导后的重组大肠杆菌上清除了DnaJ外其它5个蛋白明显表达.SDS-PAGE分析了共表达分子伴侣对玉米(Zea mays)四毗咯分子合成的谷氨酸-1-半醛氨基转移酶、尿卟琳原III脱羧酶和西罗叶绿三酸:铁鳌合酶在大肠杆菌的折叠和聚集影响,结果显示,GroEL、GroES和GrpE能防止玉米西罗叶绿三酸:铁鳌合酶在大肠杆菌的聚集,但DnaK、DnaJ和C1pB分子伴侣对该酶则没有作用;GroEL、GroES和GrpE能部分抑制玉米谷氨酸-1-半醛氨基转移酶在大肠杆菌的聚集,但是对改变玉米尿叶啉原III脱羧酶的聚集没有明显作用,这些结果表明,不同分子伴侣组对改善本研究选择的3个玉米蛋白在大肠杆菌可溶性表达的效果有差异.%The heterologous proteins are often expressed as inclusion bodies in Escherichia coli due to their inability to fold correctly.The chaperones can assist the heterologous protein folding and improve the protein aggregation in vivo.Using the E.coli strain DH5α genomic DNA as templates, six genes encoding chaperones including groEL, groES, dnaK, dnaJ, grpE and clpB were amplified respectively by PCR.The groEL, groES and grpE ( Ⅰ team) were inserted into E.coli expression plasmid pCDFDuet-1, and the dnaK, dnaJ and clpB ( Ⅱ team) were inserted into the expression plasmid pRSFDuet-1, respectively, to create the helper vector pR-GESP and pC-DJKL.Each resultant plasmid contained an artificial operon and every gene was controlled by a strong T7 promoter.The SDS-PAGE analysis showed that all proteins except DnaJ protein were expressed in the soluble extract from IPTG-induced E.coli cells containing the helper plasmid.The aggregation of maize (Zea mays) sirohydrochlorin ferrochelatase in E.coli cells was prevented by the coexpression of three chaperones GroEL,GroES and GrpE, but was not inhibited by the coexpression of the other chaperones DnaK, DnaJ and ClpB, as shown by SDS-PAGE.Overproduction of GroEL, GroES and GrpE chaperones partly suppressed the aggregation of the recombinant maize glutamate-1-semiadhyde aminotransferase, but did not function on changing the aggregation of recombinant maize uroporphyrinogen Ⅲ decarboxylase obviously, as revealed by SDS-PAGE.All results suggested that the different eharperone team had the different effects on improving the souble expression in E.coli for three maize proteins chosen in this study.