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T66I突变提高HIV-1整合酶可溶性研究
引用本文:杨东,刘斌,何红秋,张小轶,张海锋,许先进,陈慰祖,王存新.T66I突变提高HIV-1整合酶可溶性研究[J].中国生物工程杂志,2010,30(8):17-21.
作者姓名:杨东  刘斌  何红秋  张小轶  张海锋  许先进  陈慰祖  王存新
作者单位:北京工业大学生命科学与生物工程学院 北京 100124
基金项目:国家自然科学基金,北京市自然科学基金 
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。600 ml培养基中诱导表达IN1–288/T66I/BL21,亲和层析纯化共收获蛋白质4.72 mg。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。

关 键 词:整合酶  可溶性表达  T66I突变  
收稿时间:2010-03-17
修稿时间:2010-05-14

Research of the Improvement of Soluble Expression of HIV-1 Integrase Induced by T66I Mutation in E. coli
YANG Dong,LIU Bin,HE Hong-qiu,ZHANG Xiao-yi,ZHANG Hai-feng,XU Xian-jin,CHEN Wei-zu,WANG Cun-xin.Research of the Improvement of Soluble Expression of HIV-1 Integrase Induced by T66I Mutation in E. coli[J].China Biotechnology,2010,30(8):17-21.
Authors:YANG Dong  LIU Bin  HE Hong-qiu  ZHANG Xiao-yi  ZHANG Hai-feng  XU Xian-jin  CHEN Wei-zu  WANG Cun-xin
Institution:YANG Dong LIU Bin HE Hong-qiu ZHANG Xiao-yi ZHANG Hai-feng XU Xian-jin CHEN Wei-zu WANG Cun-xin (Life Science and Bio-engineering College of Beijing University of Technology,Beijing 100124,China)
Abstract: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. 4.72mg 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. 
Keywords:Integrase Soluble expression T66I mutation  
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