Purification Cholera toxin

The binding of ligands to the / -adrenergic receptor of plasma membranes stimulates adenylate cyclase activity in a process that requires GTP. The existence of a separate GTP-binding protein (42,000 daltons), besides the hormone binding component and the cyclase, was confirmed by photoaffinity labeling with y-(4-azidoanilino)-GTP (Pfeuffer, 1977). Recent purification of the GTP binding protein has confirmed the existence of a 42,000 dalton subunit that is the substrate for ADP-ribosylation by cholera toxin and NAD. 

Cholera results from an intestinal infection with the pathogenic bacterium Vibrio choleras that causes a debilitating, and even deadly, diarrhea. Successful treatment of cholera requires effective rehydration with solutions of glucose and salts (Kaper et ai, 1995). Administration of antibiotics decreases the duration of disease (Kaper et a/., 1995) vaccines are only partially effective. Koch, who first described Vibrio cholerae as the causative agent of cholera, suggested that it was a toxin-mediated disease (Koch, 1884). Over a half-century later, the existence of cholera toxin (CT) was demonstrated in cell-free culture filtrates (De, 1959 Dutta eta/., 1959) a decade later, purification of the protein toxin was achieved (Finkelstein and LoSpalluto, 1969). 

Kahn RA, Gilman AG (1984b) Purification of a protein cofactor required for ADP-ribosylation of the stimulatory regulatory component of adenylate cyclase by cholera toxin. In J. Biol. Chem. 259 6228-6234. 

Purified CTA or LTA is required for the assays described herein and can be generated and purified from CT or LT holotoxin using methods originally described for CT. Gel filtration in 0.1 M glycine buffer (pH 3.2) containing 6 M urea (Ohtomo ef al., 1976) or 5 % formic acid (Lai et al., 1976) results in separation of the A subunit from B subunit monomers. More recently, a reverse-phase HPLC procedure has been described for cholera toxin subunit purification (Pearson etal., 1986). Like CT, purified CTA can be purchased from several sources (see Section 2.2.3). 

Tayot J-L, Holmgren J, Svennerholm L, et al. (1981) Receptor-specific large-scale purification of cholera toxin on silica beads derivatized with lysoGwi ganglio-side. In Eur. J. Biochem. 113 249-258. 

Uesaka Y, Otsuka Y, Lin Z, et al. (1994) Simple method of purification of Escherichia coli heat-labile enterotoxin and cholera toxin using immobilized galactose. In Microb. Pathogen. 16 71 -76. 

Slos, P., Speck, D., Accart, N., Kolbe, H.V., Schubnel, D., Bouchon, B., Bischoff, R., and Kieny, M.P., 1994, Recombinant Cholera Toxin B Subunit in E. Coli High-level Secretion, Purification, and Characterization, In Protein Expression Purif, 5, 518-26... 

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