title: Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease.
abstract: 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.
authors.affiliation: Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
authors.forename: S K
authors.initials: SK
authors.lastname: Grant
authors.forename: I C
authors.initials: IC
authors.lastname: Deckman
authors.forename: M D
authors.initials: MD
authors.lastname: Minnich
authors.forename: J
authors.initials: J
authors.lastname: Culp
authors.forename: S
authors.initials: S
authors.lastname: Franklin
authors.forename: G B
authors.initials: GB
authors.lastname: Dreyer
authors.forename: T A
authors.initials: TA
authors.lastname: Tomaszek
authors.forename: C
authors.initials: C
authors.lastname: Debouck
authors.forename: T D
authors.initials: TD
authors.lastname: Meek
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D010450:Endopeptidases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification*; D016333:HIV Protease / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D017320:HIV Protease Inhibitors; D015497:HIV-1 / Q000201:enzymology* / Q000254:growth & development; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D011487:Protein Conformation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000145:classification / Q000302:isolation & purification; D012191:Retroviridae Proteins / Q000037:antagonists & inhibitors / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D015302:Simian Immunodeficiency Virus / Q000201:enzymology* / Q000502:physiology; D013379:Substrate Specificity
chemical_list: D017320:HIV Protease Inhibitors; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; D012191:Retroviridae Proteins; D010450:Endopeptidases; D016333:HIV Protease