Cloning Of GSH 2 Gene From Saccharomyces Cerevisiae And Construction Of Prokaryotic Expression Vector

Reduced glutathione, abbreviated as GSH, is a tri-peptide which is made up of glutamic acid, cysteine and glycine. Among these amino acids, aminoglutaminic acid'gamma carboxyl group and cysteine join into action to form peptide linkage. GSH occurs in all biological cells, and the content is higher in yeast and animal, be less in the plant. GSH plays an important role in some biological processes such as oxidation resistance,the protein sulphydryl group protection in organism,the free radical unification to protect cell, toxicity relieviation of the exogenous toxic substance and amino acids transportation. Reduced glutathione also plays important roles in plant's drought resistence and anti-heavy metal resistance. GSH acts as medical antidote to treat organic solvent, at the same time it has been widespread applied to resist oxidation and prevent skin aging. In food industry, GSH is positively exploited as health-care food for its biological activity additives.The synthesis of glutathione in yeast is catalyzed byγ-glutamyl cysteine synthetase (GSH1) and glutathione synthetase (GSH2), the GSH synthetase system in yeast and Arabidopsis thaliana etc. was cloned and analyzed one after another. Gamma glutamyl cysteine synthetase is a rate-limiting enzyme in GSH synthesis process in total organism of existing research., and it is confined by feedback inhibition of GSH, but it was discovered that glutathione synthetase is the key enzyme in GSH synthesis process in some E.coli. Moreover, it is still uncertain that whether theγ-glutamyl cysteine synthetase is a key enzyme in GSH synthesis process under certain adverse condition. For example, under Cd coercion condition, GSH1 is possibly no longer the GSH synthesis limit enzyme, because Cd coercion condition resumes GSH in plants, the feedback inhibition of GSH toγ-glutamyl cysteine synthetase is relieved.In this study, genome DNA of Saccharomyces cerevisiae was used as template, glutathione synthetase gene was amplified by polymerase chain reaction, and we constructed prokaryotic expression plasmid pET-32a (+)-gsh2 and transferred it to Escherichia coli BL21(DE3). Further, we analyzed the expression of the vector in Escherichia coli, and this research made a foundation for the further investigation of the glutathione synthetase gene of Saccharomyces cerevisiae in genetic engineering field. We expect to construct the recombinant bacteria which have high GSH synthesis activity.