Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography.

Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.