Purification, properties, and mutagenesis of poliovirus 3C protease.
Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia coli under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies. The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate. Proteolytic activity was assayed using as substrate either [35S]methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B. Poliovirus protein 3CD (protease-polymerase) was also expressed in bacteria. About 25% of this protein apparently autodigested in vivo, releasing immunoprecipitable protein 3D (polymerase). No further autodigestion of 3CD could be detected in vitro, nor could addition of purified protein 3C effect digestion in trans. Both the serine protease inhibitors PMSF, TPCK, and 3,4-dichloroisocoumarin, and the cysteine protease inhibitors cystatin and zinc, were effective inhibitors of the 3C protease. Six new mutants of the protease, with altered or no enzymatic activity, were identified based on the observation that low level expression of wild type enzyme severely retards growth of bacterial colonies harboring the expression plasmid.
Baum
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Molecular Biology Section, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965.
Bebernitz
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Palant
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Mueller
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Plotch
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D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003062:Codon; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016679:Genome, Viral; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D014764:Viral Proteins
D003062:Codon; D004279:DNA, Viral; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases