title: Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli.
abstract: Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.
authors.affiliation: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: J
authors.initials: J
authors.lastname: Boutelje
authors.forename: A R
authors.initials: AR
authors.lastname: Karlström
authors.forename: M G
authors.initials: MG
authors.lastname: Hartmanis
authors.forename: E
authors.initials: E
authors.lastname: Holmgren
authors.forename: A
authors.initials: A
authors.lastname: Sjögren
authors.forename: R L
authors.initials: RL
authors.lastname: Levine
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D016333:HIV Protease / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000138:chemical synthesis; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D015345:Oligonucleotide Probes; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D016333:HIV Protease