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Expression and characterization of a metalloprotease from a Vibrio parahaemolyticus isolate

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Abstract:

The extracellular zinc metalloprotease from Vibrio parahaemolyticus (VPM) is a putative virulence factor for host infection. It is synthesized from the vpm gene of V.parahaemolyticus as a polypeptide of 814 amino acids with an estimated molecular mass of 89833Da, containing a zinc metalloprotease HEXXH consensus motif. To investigate the enzymatic properties of V.parahaemolyticus metalloprotease, the mature vpm gene was overexpressed in Escherichia coli, and the recombinant protein (rVPM) was purified by a His-binding metal affinity column (>95% purity). The activity of the recombinant protease produced in E. coli was examined by gelatin activity staining and proteolytic activity assays using gelatin and azocasein as substrates. rVPM showed maximum activity at about 37°C and pH8. The cytotoxicity against flounder gill cells and fish pathogenicity indicated a potential role in pathogenesis.

La métalloprotéase dépendante du zinc de Vibrio parahaemolyticus (VPM) est un facteur de virulence potentiel. Elle est synthétisée à partir du gène vpm de V.parahaemolyticus sous la forme d’un polypeptide de 814 acides aminés dont la masse moléculaire est estimée à 89833Da, possédant le motif consensus HEXXH des métalloprotéases dépendantes du zinc. Afin d’examiner les propriétés enzymatiques de la métalloprotéase de V.parahaemolyticus, le gène vpm mature a été surexprimé chez Escherichia coli et la protéine recombinante (rVPM) a été purifiée à l’aide d’une chromatographie d’affinité métallique sur colonne d’His (>95% de pureté). L’activité de la protéase recombinante produite chez E. coli a été examinée par une coloration d’activité sur gélatine et par des essais d’activité protéolytique en utilisant la gélatine et l’azocaséine comme substrats. La rVPM possédait une activité maximale à 37°C environ et à pH 8. La cytotoxicité envers des cellules de branchies de carrelet et la pathogénicité chez le poisson sont l’indice d’un rôle potentiel dans la pathogenèse.

Document Type: Research Article

Publication date: October 1, 2007

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  • Published since 1954, this monthly journal contains new research in the field of microbiology including applied microbiology and biotechnology; microbial structure and function; fungi and other eucaryotic protists; infection and immunity; microbial ecology; physiology, metabolism and enzymology; and virology, genetics, and molecular biology. It also publishes review articles and notes on an occasional basis, contributed by recognized scientists worldwide.
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