Aeromonas Salmonicida Outer membrane Protein A (ompA) Recombinant Protein

Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species specific 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 protein in E. coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography yielding a 50.5 kDa monomeric protein with isoelectric point of 5.35. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western Blot techniques. All forms of A-protein were found to activate the secretion of tumour necrosis factor alpha from murine macrophage. For reference see Maurice et al. (1999) Protein Expression and Purification 16, 396-404.