Purification and characterization of HIV-1 reverse transcriptase having a 1:1 ratio of p66 and p51 subunits.
Wild-type and several mutant forms of recombinant human immunodeficiency virus type-1 reverse transcriptase were overexpressed as either the p66 or the p51 subunit in a protease-deficient strain of Escherichia coli. Immediately prior to cell lysis, p51 cell paste was mixed with cell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to the larger. During the subsequent chromatography steps stable heterodimer p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical gel filtration HPLC, laser desorption mass spectroscopy, and isoelectric focusing. In addition, we were able to obtain crystals of the purified enzyme complexed with a quinazolinone class nonnucleoside inhibitor that diffracted to 3.2 A resolution. A potential application of this expression/purification methodology is the ability to alter specific amino acids residues, by site-directed-mutagenesis, of only one subunit of the RT-dimer.
Stahlhut
M
M
Department of Biological Chemistry, Merck Research Laboratories, Division of Merck & Co., Inc., West Point, Pennsylvania 19486.
Li
Y
Y
Condra
J H
JH
Fu
J
J
Gotlib
L
L
Graham
D J
DJ
Olsen
D B
DB
D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011799:Quinazolines; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification*; D018894:Reverse Transcriptase Inhibitors
D011799:Quinazolines; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase