Expression of human glutathione S-transferase A1, P1 and T1 in Escherichia coli.
- Author:
Xiao-juan CHAI
1
,
2
;
Hai-hong HU
1
;
Lu-shan YU
1
;
Su ZENG
1
;
Author Information
- Publication Type:Journal Article
- MeSH: DNA, Complementary; genetics; Escherichia coli; genetics; Genetic Vectors; Glutathione S-Transferase pi; biosynthesis; genetics; Glutathione Transferase; biosynthesis; genetics; Humans; Recombinant Proteins; biosynthesis; Reverse Transcriptase Polymerase Chain Reaction
- From: Journal of Zhejiang University. Medical sciences 2014;43(2):168-174
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo construct the vectors of human glutathione S-transferase A1 (GSTA1), P1 (GSTP1), T1(GSTT1) genes and express in Escherichia coli (E. coli).
METHODSHuman 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.
RESULTSThe 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.
CONCLUSIONThe expression plasmids for GSTA1, GSTT1 and GSTP1 have been constructed and the recombinant proteins are expressed successfully.
