Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease

The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 1...

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Bibliographic Details
Published in:The Journal of biological chemistry 1989-02, Vol.264 (4), p.2307-2312
Main Authors: Darke, P L, Leu, C T, Davis, L J, Heimbach, J C, Diehl, R E, Hill, W S, Dixon, R A, Sigal, I S
Format: Article
Language:English
Subjects:
AIDS/HIV
Aspartic Acid Endopeptidases
Biological and medical sciences
Biotechnology
Chromatography, Ion Exchange
Cloning, Molecular
DNA, Recombinant - metabolism
Endopeptidases - genetics
Endopeptidases - isolation & purification
Endopeptidases - metabolism
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gene expression
Genes
Genes, Viral
Genetic engineering
Genetic technics
HIV-1 - enzymology
HIV-1 - genetics
human immunodeficiency virus
Immunoblotting
Kinetics
Methods. Procedures. Technologies
Molecular and cellular biology
Molecular cloning
Molecular genetics
Molecular Weight
Plasmids
Substrate Specificity
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Summary:The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.
ISSN:0021-9258
1083-351X