Cloning and expression of γ-glutamylcysteine synthetase from Saccharomyce cerevisiae and structure analysis

The γ-glutamylcysteine synthetase ( GSH Ⅰ) is the key enzyme for biosynthesis of glutathione.Construction of one recombinant bacteria for GSH Ⅰ with higher activity would improve its glutathione synthesis capacity. The gene encoding GSH Ⅰ was amplified from Saccharomyces cerevisiae 2-10515 by using PCR technique.The cloned gene was inserted into an expression vector pET-28a to produce the recombinant expression plasmid pET-28a-gsh Ⅰ, and then transformed into E.coli BL21 (DE3) . After IPTG induction, the activity of GSH Ⅰ of the engineered strain reached 46.09U/ (mg the wet cell) . The sequence of gsh Ⅰ gene and the structure of GSH Ⅰ protein were analyzed and predicted with bioinformatics.This provided theory evidence for construction of recombinant bacteria for GSH Ⅰ with high level and activity.