Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease

• Published in: Biochemistry (Easton) 1991-08, Vol.30 (34), p.8424-8434
• Main Authors: Grant, Stephan K, Deckman, Ingrid C, Minnich, Michael D, Culp, Jeffrey, Franklin, Samuel, Dreyer, Geoffrey B, Tomaszek, Thaddeus A, Debouck, Christine, Meek, Thomas D
• Format: Article
• Language: English
• Subjects: AIDS/HIV; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Endopeptidases - chemistry; Endopeptidases - classification; Endopeptidases - isolation & purification; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; HIV Protease - classification; HIV Protease - genetics; HIV Protease - isolation & purification; HIV Protease Inhibitors; HIV-1 - enzymology; HIV-1 - growth & development; human immunodeficiency virus 1; Hydrogen-Ion Concentration; Hydrolases; Isoelectric Focusing; Molecular Sequence Data; Peptides - chemical synthesis; Peptides - pharmacology; Protein Conformation; Protein Synthesis Inhibitors - pharmacology; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - biosynthesis; Recombinant Proteins - classification; Recombinant Proteins - isolation & purification; Retroviridae Proteins - antagonists & inhibitors; Retroviridae Proteins - classification; Retroviridae Proteins - genetics; Retroviridae Proteins - isolation & purification; simian immunodeficiency virus; Simian Immunodeficiency Virus - enzymology; Simian Immunodeficiency Virus - physiology; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00098a021

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.