酿酒酵母GSH Ⅰ基因的克隆、表达与结构分析

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

摘要: γ-谷氨酰半胱氨酸合成酶（GSHI）是合成谷胱甘肽的关键酶。通过构建GSHI活性较高的重组菌来提高合成GSH的能力。利用PCR技术扩增获得了酿酒酵母（Saccharomyces cerevisiae）2-10515的gshI基因,构建原核表达载体pET-28a-gshI,转化到Escherichia coli BL21（DE3）中,重组菌经诱导表达后,测得GSHI的酶活力为46.09U/mg湿菌,活性较高。进一步利用生物信息学方法分析和预测GSHI基因的序列和蛋白结构,为在基因水平上提高该酶的表达量和活性提供了理论依据。

Abstract: 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.