Certificate of Analysis & Data Sheet

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.

E. coli

White lyophilized (freeze-dried) powder

The protein was lyophilized from a concentrated (1mg/ml) solution with 0.02% NaHCO3.

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.

Lyophilized A-protein although stable at room temperature for several weeks, should be stored desiccated below -18 C. Upon reconstitution at > 0.1 A-protein mg/ml and up to 2 mg and filter sterilization A-protein can be stored at +4C

Greater than 98.0% as determined by:
(a) Gel filtration analysis.
(b) Analysis by reducing and non-reducing SDS-PAGE gel.
(c) Gel-filtration chromatography under non denaturing conditions.

The N-terminal amino sequence is Met-Asp-Val-Val-Ile-Ser

Less than 3 %

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%.

Less than 0.1 ng/µg (IEU/µg) of A-protein

Protein quantitation was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.58 as the extinction coefficient for a 0.1% (1mg/ml) solution at pH 8.0. This value is calculated by the PC GENE computer analysis program of protein sequences (IntelliGenetics).

This material is offered by PLR for laboratory research ONLY