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

• Published in: FEBS letters Vol. 280; no. 2; pp. 347 - 350
• Main Authors: Wondrak, Ewald M., Louis, John M., Mora, Peter T., Oroszlan, Stephen
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 25.03.1991; Elsevier
• Subjects: affinity chromatography; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Affinity; Endopeptidases; Enzymes and enzyme inhibitors; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; HIV Protease - genetics; HIV Protease - isolation & purification; HIV-1 - enzymology; HIV-1 - genetics; HIV-1 protease; Hydrolases; inhibitors; Molecular Sequence Data; Mutant protease; Mutation; Pepstatin A affinity chromatography; Pepstatins; proteinase; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Mutant protease; HIV-1 protease; Pepstatin A affinity chromatography; Virus; Characterization; Purification; HIV-1 virus; Affinity chromatography; Enzyme; Proteinase; Retroviridae; Lentivirinae; Human immunodeficiency virus; Mutation
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(91)80328-Z

Recombinant wild-type protease of human immunodeficiency virus, type [(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/μg. Two proteolytically inactive HIV-1 mutant proteases (Arg-87 → Lys; Asn-88 → Glu) were found to bind to pepstatin A agarose, 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.