Buitenmembraaneiwit A (ompA)

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.

Outer membrane protein assembly factor BamE

79880640

A4SPV3

WP_011898937.1

Aeromonas Salmonicida

It is recommended to reconstitute the lyophilized A-protein in sterile 0.4% NaHCO3 adjusted, not less than 100µg/ml, which can then be further diluted to other aqueous solutions.

< 0.1 ng/µg of protein (< 1EU/µg)

>98.0% as determined by Gel filtration analysis and SDS-PAGE gel.

The interaction of bacterial and recombinant A-layer protein with murine macrophages was directed at determining the effect of A-protein on intracellular events that occur in primed macrophages. This was accomplished by measuring the cytotoxic product produced by peritoneal macrophages when exposed to A-protein coated latex beads. Thioglycolate elicited macrophages exhibited a low level of activation (18% cytotoxicity) that was significantly increased (48% cytotoxicity) in the presence of latex beads. Coating of the latex beads with each of the three A-protein products resulted in an increase of cytoxicity (mean +/- SEM) from 48% to 91%.

Lyophilized

0.4% NaHCO3, pH 8-9

50.5 kDa

E. coli

MDVVIS