FORMATION OF HETERODIMERS OF HUMAN-IMMUNODEFICIENCY-VIRUS-TYPE-1 REVERSE-TRANSCRIPTASE BY RECOMBINATION OF SEPARATELY PURIFIED SUBUNITS

Human-immunodeficiency-virus-type-1 reverse transcriptase exists in virions as a heterodimer of a M(r) 66000 subunit and its C-terminally truncated form of M(r) 51000, but, when expressed as a recombinant M(r) 66000 protein, a mixture of heterodimers and homodimers results which co-purify by most conventional techniques. We describe a method of hydrophobic chromatography which gives baseline separation of these two forms of the protein. This method has been applied to purify heterodimers formed by recombination of separately expressed and purified M(r) 66000 and 51000 subunits, resulting in significantly more homogeneous heterodimer preparations. The recombined heterodimer showed similar kinetic properties and RNase H activity to the standard heterodimer and a specific activity significantly higher than the original homodimer of the M(r) 66000 protein. Heterodimers having greater asymmetry have also been prepared by recombining M(r) 66000 subunits containing single-point or deletion mutations, with wild-type M(r) 51000 subunits, and the resulting heterodimers analysed.