Isolation and properties of adenovirus type 2 proteinase.
We have cloned and expressed the human adenovirus type 2 proteinase gene in Escherichia coli. The expressed proteinase was isolated by a four-step chromatographic procedure. Purity and identity of the recombinant protein was established by two-dimensional gel electrophoresis, N-terminal sequencing, and specific antisera. The pure enzyme did not contain disulfide bridges, and it consisted of one subunit with a pI of 10.2. It did not show any sign of autocleavage. Labeled iodoacetate bound the pure enzyme while labeled diisopropyl fluorophosphate did not. The protease readily cleaved the viral pVII protein, ovalbumin, fibrin, and actin but had no effect on synthetic penta-, octa-, or nonapeptides carrying the consensus sequence for cleavage. The inhibitory profile of the isolated proteinase and the affinity labeling clearly indicate that the human adenovirus type 2 proteinase is a cysteine rather than a serine proteinase as previously believed. The most likely candidate for an active site residue is one of the two conserved cysteines, Cys-104 or Cys-122.
Department of Microbiology, Faculty of Medicine, University of Sherbrooke, Québec, Canada.
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Tihanyi
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Bourbonnière
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Houde
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Rancourt
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JM
Weber
D000260:Adenoviruses, Human / Q000201:enzymology*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D011487:Protein Conformation; D012995:Solubility; D013379:Substrate Specificity
D000345:Affinity Labels; D004220:Disulfides; D009842:Oligopeptides; D010446:Peptide Fragments; D004262:DNA Restriction Enzymes; D003546:Cysteine Endopeptidases; C091827:adenain