Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities.
Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities.
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重组 HIV-1 逆转录酶：纯化、一级结构和聚合酶/核糖核酸酶 H 活性。
DOI：
10.1016/0003-9861(89)90493-1
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发表时间：
1989
影响因子：
3.9
通讯作者：
Debouck,C
中科院分区：
文献类型：
--
作者：
Mizrahi,V;Lazarus,GM;Miles,LM;Meyers,CA;Debouck,C
重组HIV-1逆转录酶（RT）作为可溶性蛋白在大肠杆菌中稳定过量产生，使用双质粒表达系统，在该系统中，RT前体蛋白被HIV-1蛋白酶产生的内含子在体内表达和加工。由此产生的RT由p66和p51两个多肽组成，其同源性为90%，通过多肽混合物的序列分析发现它们具有共同的nh2末端。观察到的序列证实了蛋白酶在蛋白酶-RT多蛋白连接处进行的内切校正，产生与真正的病毒RT相同的nh2末端(M.M. Lightfooteet al.(1986)J。Virol.60, 771 - 775;F. diMarzo Veroneseet al.（1986）《科学》231,1289-1291)。细菌表达的RT具有与病毒RT相似的特异性活性，用膦甲酸酯进行的抑制研究证实，在对该抑制剂的敏感性方面，它与病毒酶没有区别。聚合酶活化凝胶分析表明，p66比p51具有更高水平的RT活性。RNase H活化凝胶分析表明，纯化后的重组RT不含内源性ouse。RT的RNase H活性仅与p66多肽相关，支持RNase H结构域位于分子cooh末端区域的假设。
Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein inEscherichia coliusing a double-plasmid expression system in which an RT precursor protein was expressed and processedin vivoby HIV-1 protease produced intrans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to >90% homogeneity and were found to share a common NH2terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correctin vivocleavage by protease at the protease-RT polyprotein junction to yield an NH2terminus identical to that of genuine viral RT (M.M. Lightfooteet al.(1986)J. Virol.60, 771–775; F. diMarzo Veroneseet al.(1986)Science231, 1289–1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenousE. coliRNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.