Production of Vibrio cholerae accessory cholera enterotoxin (Ace) in the yeast Pichia pastoris

Abstract

Accessory cholera enterotoxin (Ace) is a recently identified toxin of Vibrio cholerae. Preliminary studies using crude toxin extracts in animal models indicate that Ace increases transcellular ion transport, which is proposed to contribute to diarrhea in cholera. The lack of purified toxin has hindered elucidation of the mechanism of action of Ace. In this study, ace was cloned and was expressed in and secreted by the methylotrophic yeast Pichia pastoris. Secreted toxin constituted 50% of the total supernatant protein from Pichia pastoris. Presumed monomer and dimer forms with molecular masses of 9 and 18 kDa, respectively, were observed. The 18-kDa form predominated. Biological activity was assayed by studying ion fluxes across epithelial membranes in Ussing chambers. Among the characteristics of Ace was the unusual property of staining with silver but not Coomassie blue stain. To our knowledge this is the first report of a biologically active bacterial toxin produced with the P. pastoris system. The purified protein may now be used in studies of the mechanism of action of Ace in physiologic systems.