[谷胱甘肽合成酶基因的克隆与表达]

[Cloning and expression of the genes of glutathione synthetases].

作者信息

Shen L X, Wei D Z, Zhao Z F, Zhang S L, Wang E L

机构信息

State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science & Technology, Shanghai 200237, China.

出版信息

Sheng Wu Gong Cheng Xue Bao. 2001 Jan;17(1):98-100.

PMID:11330198

Abstract

The genes(gsh-I,gsh-II) for gamma-glutamyl-cysteine synthetase(GSH-I) and glutathione synthetase(GSH-II) from Escherichia coli B were amplified by PCR and then subcloned into plasmid pUC19 respectively. The DNA fragments harboring gshII and gsh I were inserted into plasmid pTrc99A one by one to get a hybrid plasmid pTrc-gsh. E. coli BL21 was transformed by pTrc-gsh for expression of the related enzymes. Analysis of SDS-PAGE showed that the expected products were expressed. E. coli BL21(pTrc-gsh) were incubated at 37 degrees C and pH 7.2 to OD550 = 0.5. The conditions were then switched to 34 degrees C and pH6.7 after the addition of 0.1 mmol/L IPTG. The expressed products were up to 25% of the total protein of the bacteria. Acetone-treated cells of the engineered strain could synthesize GSH efficiently.

摘要

通过PCR扩增来自大肠杆菌B的γ-谷氨酰半胱氨酸合成酶（GSH-I）和谷胱甘肽合成酶（GSH-II）的基因（gsh-I、gsh-II），然后分别亚克隆到质粒pUC19中。将携带gshII和gsh I的DNA片段逐一插入质粒pTrc99A中，得到杂交质粒pTrc-gsh。用pTrc-gsh转化大肠杆菌BL21以表达相关酶。SDS-PAGE分析表明表达出了预期产物。将大肠杆菌BL21(pTrc-gsh)在37℃和pH 7.2下培养至OD550 = 0.5。添加0.1 mmol/L IPTG后，条件转换为34℃和pH6.7。表达产物高达细菌总蛋白的25%。工程菌株经丙酮处理的细胞能够高效合成谷胱甘肽。