Stability study of Schistosoma japonicum glutathione-S-transferse and its site-directed mutants

• Main Author: 羅森林
• Other Authors: 陳秀美
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/95325711541388098739

碩士 === 國立臺灣科技大學 === 化學工程技術研究所 === 86 === 　　A Schistosoma japonicum glutathione S-transferase (GST) and its four different mutants were studied to investigate the effect of a C-terminal addition of a metal affinity tag, 6His Tag, on the stability of the enzyme and the effects of cysteine (Cys)oxidation on the activity and the stability of the one. These mutants are GST/His (a fusion protein of GST and 6 His Tag), Cys85→Ser GST/His (a GST/His mutant where Cys 85 was replaced with Ser), Cys138→Ser GST/His and Cys 178→Ser GST His. The results showed that: 　　(1)GST/His expressed using T7 promoter was better produced than GST expressed using tac promoter. 　　(2)For GST/His and its mutants, the purification with Ni2+-NTA gels had better stability and application than the purification with glutathione (GSH) Sepharose 4B gels. 　　(3)Without the presence of reducing agents, the structures of GST and GST/His could be changed due to the oxidation of their Cys residues. 　　(4)The results of the kinetic studies showed that the Cys 85 and Cys 138 residues on GST/His had some contributions to the function and the structure of the G-site and the H-site of the enzyme, respectively. 　　(5)Under a denaturing condition where denaturant existed, the structures of GST and GST/His underwent reversible changes due to unfolding. 　　(6)At higt denaturant concentrations, GSH could protect GST and GST/His from strucrural changes and inactivation due to unfolding. 　　(7)The thermoinactivation rates of GST/His and its mutants were reduced when they were immobilized on Ni2+-NTA gels.