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Purification of HIV‐1 wild‐type protease and characterization of proteolytically inactive HIV‐1 protease mutants by pepstatin A affinity chromatography
Author(s) -
Wondrak Ewald M.,
Louis John M.,
Mora Peter T.,
Oroszlan Stephen
Publication year - 1991
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(91)80328-z
Subject(s) - pepstatin , protease , proteases , affinity chromatography , biochemistry , mutant , wild type , biology , indinavir , enzyme , recombinant dna , hiv 1 protease , saquinavir , microbiology and biotechnology , chemistry , human immunodeficiency virus (hiv) , virology , sida , viral disease , antiretroviral therapy , viral load , gene
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.