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.
Wondrak
E M
EM
Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research and Development Center, MD 21702.
Louis
J M
JM
Mora
P T
PT
Oroszlan
S
S
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D016333:HIV Protease / Q000235:genetics / Q000302:isolation & purification*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D010436:Pepstatins; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
D010436:Pepstatins; D011994:Recombinant Proteins; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016333:HIV Protease; C031375:pepstatin