Trucksis M, Conn T L, Fasano A, Kaper J B
Department of Medicine, University of Maryland School of Medicine, and Medical Science, Veterans Affairs Medical Center, Baltimore 21201, USA.
Infect Immun. 1997 Dec;65(12):4984-8. doi: 10.1128/iai.65.12.4984-4988.1997.
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.
霍乱弧菌附属肠毒素（Ace）是一种最近才被鉴定出来的霍乱弧菌毒素。在动物模型中使用粗毒素提取物进行的初步研究表明，Ace会增加跨细胞离子转运，这被认为是导致霍乱腹泻的原因之一。缺乏纯化毒素阻碍了对Ace作用机制的阐明。在本研究中，ace基因被克隆，并在甲基营养型酵母毕赤酵母中表达和分泌。分泌的毒素占毕赤酵母总上清蛋白的50%。观察到推测的单体和二聚体形式，分子量分别为9 kDa和18 kDa。18 kDa的形式占主导。通过研究Ussing室中上皮细胞膜的离子通量来测定生物活性。Ace的特点之一是具有用银染色而非考马斯亮蓝染色的不寻常特性。据我们所知，这是关于用毕赤酵母系统生产生物活性细菌毒素的首次报道。现在，纯化的蛋白质可用于研究Ace在生理系统中的作用机制。
