Mizrahi V, Lazarus G M, Miles L M, Meyers C A, Debouck C
Department of Macromolecular Sciences, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Arch Biochem Biophys. 1989 Sep;273(2):347-58. doi: 10.1016/0003-9861(89)90493-1.
Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 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 endogenous E. coli RNase 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.
使用双质粒表达系统在大肠杆菌中稳定过量表达重组HIV-1逆转录酶（RT），使其作为可溶性蛋白表达。在该系统中，RT前体蛋白在体内由反式表达的HIV-1蛋白酶进行表达和加工。由此产生的RT由两种多肽p66和p51的等摩尔混合物组成，它们经共纯化后纯度大于90%，通过对多肽混合物的序列分析判断，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多蛋白连接处的体内切割是正确的，从而产生了与真正病毒RT相同的NH2末端（M.M.Lightfoote等人（1986年）《病毒学杂志》60卷，771-775页；F.diMarzo Veronese等人（1986年）《科学》231卷，1289-1291页）。细菌表达的RT具有与病毒RT相似的比活性，用膦甲酸进行的抑制研究证实，就对该抑制剂的敏感性而言，它与病毒酶没有区别。对混合物进行的聚合酶激活凝胶分析表明，p66的RT活性水平高于p51。核糖核酸酶H激活凝胶分析表明，重组RT的纯化制剂不含内源性大肠杆菌核糖核酸酶H，并且RT的核糖核酸酶H活性仅与p66多肽相关，这支持了核糖核酸酶H结构域位于分子COOH末端区域的假设。
