A-protein from achromogenic atypical Aeromonas salmonicida: Molecular cloning, expression, purification, and characterization

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldf ish ulcer disease. Virulence of this bacterium is associated with the produ ction of a paracrystalline outer membrane A-layer protein. The species-spec ific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the pr otein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and puri fied by ion exchange chromatography on Q-Sepharose to over 95% pure monomer ic protein. Recombinant A-protein was compared by biochemical, immunologica l, and molecular methods with the A-protein isolated from atypical A. salmo nicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type w hen examined by SDS-PAGE and gel filtration chromatography, The immunologic al similarity of the protein samples was demonstrated by employing polyclon al and monoclonal antibodies in ELISA and Western blot techniques. All form s of A-protein were found to activate the secretion of tumor necrosis facto r cu from murine macrophage, To date, this represents the first large-scale production of biologically active recombinant A-protein. (C) 1999 Academic Press.