[Expression and activity analysis of the human glutathione-S-transferase in Lactococcus lactis].
The glutathione-S-transferase A1 cDNA was amplified from human liver total RNAs by RT-PCR and was cloned into a Escherichia coli expression vector pET23b, then the recombinant plasmid pET23bhgst was introduced into E. coli BL21 (DE3) and induced by IPTG, the high-level expression of hGSTA1 appeared in the E. coli cells. The cDNA encoding hGSTA1 was subcloned into pMG36e, a lactococcal expression vector, and introduced into Lactococcus lactis MG1363 by electroporation. In the positive transformants, the hGSTA1 was expressed as a fusion protein which was verified by SDS-PAGE and Western blot. The hGSTA1 from both E. coli and L. lactis was purified by affinity chromatography on glutathione-agarose and all showed enzymatic activity. The potential application of the recombinant Lactococcus lactis in functional food was also discussed.
Xiang
H
H
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080.
Xhang
Y
Y
Wei
W
W
Tan
H
H
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013294:Lactococcus lactis / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D010957:Plasmids; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005982:Glutathione Transferase