title: Purification of turnip mosaic potyvirus viral protein genome-linked proteinase expressed in Escherichia coli and development of a quantitative assay for proteolytic activity.
abstract: The 49-kDa, nuclear inclusion a-like, viral protein genome-linked proteinase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VPg-Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N-terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capable of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-His-Gln-Ala-Ala-NH2, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gln and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPg-Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH-activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.
authors.affiliation: Biotechnology Research Institute, National Research Council Canada, Québec.
authors.forename: R
authors.initials: R
authors.lastname: Ménard
authors.forename: H
authors.initials: H
authors.lastname: Chatel
authors.forename: R
authors.initials: R
authors.lastname: Dupras
authors.forename: C
authors.initials: C
authors.lastname: Plouffe
authors.forename: J F
authors.initials: JF
authors.lastname: Laliberté
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D010944:Plants / Q000821:virology; D017800:Potyvirus / Q000201:enzymology*; D013379:Substrate Specificity; D014758:Viral Core Proteins / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D018076:DNA, Complementary; D010455:Peptides; D014758:Viral Core Proteins; D010450:Endopeptidases