Gerhard Ralf, Burger Silke, Tatge Helma, Genth Harald, Just Ingo, Hofmann Fred
Institute of Toxicology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Microb Pathog. 2005 Feb-Mar;38(2-3):77-83. doi: 10.1016/j.micpath.2004.12.002.
Toxins A and B from Clostridium difficile are single-chain proteins of 308,000 and 270,000 Da, respectively. They possess transferase activity to monoglucosylate proteins of the Rho GTPase family whereby Rho, Rac, and Cdc42 are the canonical substrates. For application of these toxins as specific Rho GTPase inhibitors the highest possible purity is of crucial interest. We, therefore, expressed recombinant His-tagged toxin A using the Bacillus megaterium expression system. Specific antisera raised against the native toxin A from C. difficile and the recombinant toxin, respectively, showed identical sensitivity and specificity in Western blot and ELISA analyses towards both toxins. By comparison of both toxins in functional studies we showed that the recombinant toxin was about two times more cytotoxic than the native toxin, and the glucosyltransferase-activity of the recombinant toxin was even 10-fold increased. However, recombinant toxin A showed one essential difference to the classically purified one. The reported transferase-independent effect of toxin A to release cytochrome c from isolated mitochondria was not exhibited by the recombinant toxin A. This putative mitochondrial effect decreased with increased purity of toxin A, and was absent with recombinant toxin, strongly suggesting an clostridial contamination responsible. In summary, we tested the recombinant toxin A to be at least an adequate substitute for the native toxin, bearing the advantage of a rapid single-step purification and the absence of biological active contaminations.
艰难梭菌的毒素A和毒素B分别是分子量为308,000道尔顿和270,000道尔顿的单链蛋白质。它们具有将Rho GTPase家族的蛋白质单糖基化的转移酶活性，其中Rho、Rac和Cdc42是典型的底物。对于将这些毒素用作特异性Rho GTPase抑制剂而言，尽可能高的纯度至关重要。因此，我们使用巨大芽孢杆菌表达系统表达了重组His标签毒素A。分别针对来自艰难梭菌的天然毒素A和重组毒素制备的特异性抗血清，在蛋白质印迹和酶联免疫吸附测定分析中对两种毒素显示出相同的敏感性和特异性。通过在功能研究中比较两种毒素，我们发现重组毒素的细胞毒性比天然毒素高约两倍，并且重组毒素的葡萄糖基转移酶活性甚至提高了10倍。然而，重组毒素A与经典纯化的毒素A表现出一个重要差异。重组毒素A未表现出毒素A从分离的线粒体中释放细胞色素c的报道的非转移酶依赖性效应。这种假定的线粒体效应随着毒素A纯度的提高而降低，并且在重组毒素中不存在，这强烈表明是梭菌污染所致。总之，我们测试了重组毒素A至少是天然毒素的合适替代品，具有快速一步纯化且不存在生物活性污染物的优点。
