T66I突变提高HIV-1整合酶大肠杆菌表达可溶性研究  

Research of the Improvement of Soluble Expression of HIV-1 Integrase Induced by T66I Mutation in E.coli

作　　者：杨东[1] 刘斌[1] 何红秋[1] 张小轶[1] 张海锋[1] 许先进[1] 陈慰祖[1] 王存新[1] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124

出　　处：《中国生物工程杂志》2010年第8期17-21,共5页China Biotechnology

基　　金：国家自然科学基金(30670497);北京市自然科学基金(7082006)资助项目

摘　　要：在研究HIV-1整合酶(IN)抗药性突变T66I时,发现这一突变同时可以提高整合酶的溶解性。原核表达了IN1?288/T66I和野生型(WT),取菌体破碎后的上清,SDS-PAGE和his标签蛋白质染色进行分析,结果表明IN1?288/T66I可溶性约是WT的2.4倍。600ml培养基中诱导表达IN1?288/T66I/BL21,亲和层析纯化共收获蛋白质4.72mg。用改进的ELISA方法测定IN1?288/T66I和IN1-288/F185K/C280S链转移催化活性,结果显示两种蛋白质活性基本相当。提供了有别于F185K/C280S突变的另外一种整合酶可溶性表达的途径,IN1?288/T66I重组蛋白还可以应用到整合酶抑制剂筛选中,以获取避开T66I抗药性突变的抑制剂。During research of the reported drug-resistance mutation T66I, it was found that this mutation can also improve the solubility of integrase. IN1 -288/T66I and WT were expressed at the same time, 10μl of the supernatant was extracted for SDS-PAGE analysis and His-tagged protein staining. Result shows that the solubility of IN1-288/T66I is 2 - 3 fold of WT. 4.72rag protein was obtained from 600ml of cells expressing IN1-288/T66I. The strand transfer activity of IN1-288/T66I and IN1-288/F185K/C280S were analyzed by advanced ELISA, which shows that two proteins are almost equal in catalytic activity. Another method, being different from F185K / C280S mutation, to improve the soluble expression of integrase has been provided. IN1-288/T66I can also be used to screen integrase inhibitor, which can avoid the drug-resistance brought by T66I mutation.

关 键 词：整合酶 可溶性表达 T66I突变 

分 类 号：Q819[生物学—生物工程]