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 immunodeficien cy 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 c ell paste containing the corresponding overexpressed p66 subunit in a ratio resulting in an excess of the smaller subunit with respect to th e larger. During the subsequent chromatography steps stable heterodime r p66/p51 was purified to homogeneity. This protein was characterized by amino acid analysis, denaturing sodium dodecyl sulfate-polyacrylami de gel electrophoresis, analytical gel filtration HPLC, laser desorpti on mass spectroscopy, and isoelectric focusing. In addition, we were a ble to obtain crystals of the purified enzyme complexed with a quinazo linone class nonnucleoside inhibitor that diffracted to 3.2 Angstrom r esolution. A potential application of this expression/purification met hodology is the ability to alter specific amino acids residues, by sit e-directed-mutagenesis, of only one subunit of the RT-dimer. (C) 1994 Academic Press, Inc.