农业生物技术学报2011，Vol.19Issue(1)：171-177,7.DOI:10.3969/j.issn.1674-7968.2011.01.025
大肠杆菌分子伴侣表达的辅助载体构建及应
The Helper Vectors Construction of Chaperones from Escherichia coli and Their Application
摘要
Abstract
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.关键词
分子伴侣/大肠杆菌/辅助载体/构建/应用Key words
Chaperone/ Escherichia coli/ Helper vector/ Construction/ Application引用本文复制引用
亓振国,张宽亮,陈宗梅,程备久,范军..大肠杆菌分子伴侣表达的辅助载体构建及应[J].农业生物技术学报,2011,19(1):171-177,7.基金项目
本研究由转基因生物新品种培育重大专项(No.2009ZX0810-002B)、国家自然科学基金(No.30840018)和安徽省攻关课题((No.0701302137)共同资助 （No.2009ZX0810-002B）
