Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities.
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.
Mizrahi
V
V
Department of Macromolecular Sciences, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406.
Lazarus
G M
GM
Miles
L M
LM
Meyers
C A
CA
Debouck
C
C
D000596:Amino Acids / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004722:Endoribonucleases / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D015497:HIV-1 / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H
D000596:Amino Acids; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H