gsh 1基因的克隆及其在巴斯德毕赤氏酵母中的表达  

Cloning and Expressing of gsh 1 gene in Pichia pastoris

作　　者：饶志明[1] 艾丽静[1] 沈微[1] 方慧英[1] 诸葛健[1] 

机构地区：[1]江南大学工业微生物中心和工业生物技术教育部重点实验室,江苏无锡214036

出　　处：《应用与环境生物学报》2007年第2期257-260,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家自然科学基金(No.30570142);江苏省青年科技创新人才(学术带头人)基金(BK2006504);教育部长江学者和创新团队发展计划;江南大学工业生物技术教育部重点实验室开放课题(KLIB-KF-200507)资助~~

摘　　要：利用PCR技术克隆了来源于Saccharomyces cerevisiae ALJ036的γ-L-谷氨酰-L-半胱氨酸合成酶(γ-glutamyl-cysteine synthetase)编码基因gsh1,连接多拷贝表达载体pGAPZA,转化Pichia pastorisx-33,构建了重组菌P.pastorisx-33(pGAPZA-gsh1);在高浓度Zeocin平板上筛选多拷贝阳性转化子,并通过PCR进行了验证.对阳性转化子的γ-L-谷氨酰-L-半胱氨酸合成酶酶活力进行了测定,结果显示,重组酵母的γ-L-谷氨酰-L-半胱氨酸合成酶的酶活是宿主菌的3.0倍,在液体YEPD中重组菌GSH的产量为42.2mg/L,是宿主菌的1.6倍.The gene encoding y-glutamyl-cysteine synthetase （gsh 1 ） was cloned to vector pGAPZA and then transformed into Pichia pastoris x-33 strain after lineafized by Avr Ⅱ. Recombinants resistant to zeocin were selected and identified by PCR analysis. The selected transformants were cultivated in flasks. The activity of γ-glutamyl-cysteine synthetase in the recombinant P. pastoris x-33 （pGAPZA-gsh 1） was 98.5 U/mg protein, which was 3 times as that of the host strain, while the yield of GSH in the recombinant P. pastoris x-33 （ pGAPZA-gsh 1 ） was 42.2 mg/L, 1.6 times as that of P. pastoris x-33. Fig 7, Tab 1, Ref 10

关 键 词：GSH 1 巴斯德毕赤氏酵母 克隆 表达 谷胱甘肽 γ- 谷氨酰半胱氨酸合成酶 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]