Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli
10.3785/j.issn.1008-9292.2014.03.009
- VernacularTitle:人谷胱甘肽硫转移酶的克隆表达及活性鉴定
- Author:
Xiao-Juan CHAI
1
;
Hai-Hong HU
;
Lu-Shan YU
;
Su ZENG
Author Information
1. 浙江大学药学院药物分析与药物代谢研究室浙江省抗肿瘤药物临床前研究重点实验室
- Keywords:
Glutathione transferase /biosynthesis;
Cloning,molecular;
Gene expression;
Chromatography,affinity;
Gene amplification;
Plasmids;
Reverse transcriptase polymerase chain reaction
- From:
Journal of Zhejiang University. Medical sciences
2014;(2):168-174
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli).Methods: Human GSTA1, GSTP1 and GSTT1 gene whole length cDNAs were amplified by RT-PCR and then subcloned into pET-28a(+) vectors.The proteins were expressed in E.coli BL21(DE3).After purified by Ni2+affinity chromatography, the enzymatic activities of GSTs were measured with 1-chloro-2,4 -dinitrobenzene (CDNB) as substrate .Results:The correct GSTA1, GSTP1 and GSTT1 genes were cloned .And soluble GSTA1, GSTT1, GSTP1 proteins were expressed in E.coli.After purification, GSTA1, GSTT1 and GSTP1 showed good enzymatic activities , which were 17.55, 0.02, 18.75 μmol· min-1 · mg-1 , respectively.Conclusion: The expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully .
