Expression and purification of active form of HIV-1 protease from E.coli.
We have subcloned an N-terminal extended protease gene of human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame into expression vector pGEX-KG. A relatively high level of expression of recombinant HIV-1 protease (PR) was achieved with isopropyl beta-D-thiogalactoside (IPTG) induction and glucose supplement. An isolation method consisting of denaturation of protein and followed by refolding was developed for releasing this recombinant HIV-1 PR into the soluble phase since most of the expressed protease was initially present in insoluble inclusion bodies. High purity of this recombinant HIV-1 PR was obtained by sequential purification using Sephadex G-50 gel filtration and CM-23 cellulose cation exchange chromatography, yielding the protease more than 1 mg per liter culture. N-terminal amino acid sequence analysis showed that the recombinant HIV-1 PR underwent autocleavage from the fusion protein during expression. SDS-PAGE indicated that the molecular weight of this recombinant HIV-1 PR is 11 kDa. This recombinant HIV-1 PR showed proteolytic activity for the synthetic peptide substrates corresponding to the sequence at the Gag MA/CA and Pol p6*/PR junctions. The purified enzyme whose specific activity for the heptapeptide SQNYPIV was 848.7 nmol*min-1*mg protease-1 also processed recombinant polyprotein Gag41 as its substrate.
Wan
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M
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Kent Ridge Crescent.
Loh
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D015967:Gene Expression Regulation, Viral; D015750:Genes, gag / Q000235:genetics / Q000502:physiology; D015751:Genes, pol / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors; D005947:Glucose / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics; D016333:HIV Protease / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015497:HIV-1 / Q000201:enzymology*; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D005982:Glutathione Transferase; D010447:Peptide Hydrolases; D016333:HIV Protease; D005947:Glucose