Gad Wael, Ben-Abderrazek Rahma, Wahni Khadija, Vertommen Didier, Muyldermans Serge, Bouhaouala-Zahar Balkiss, Messens Joris
§Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis-Université Tunis El Manar, Tunisia.
∥de Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium.
Biosci Rep. 2014 Jul 29;34(4):e00122. doi: 10.1042/BSR20140050.
Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD50 (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice.
蝎子蜇伤后的中毒是世界许多地区常见的紧急情况。在蝎子蜇伤中毒过程中，毒液中剧毒的小多肽在受害者体内迅速扩散，引发严重的医学问题。探索毒素的结构 - 功能关系将受益于在大肠杆菌中产生可溶性重组蝎子毒素。我们开发了一种体外小麦胚翻译系统，用于表达需要正确形成四个二硫键的剧毒Aah（澳大利亚杀人蝎）II蛋白。在该体外翻译系统中获得了可溶性的、重组的N端带有GST（谷胱甘肽S - 转移酶）标签的AahII毒素。在通过蛋白酶去除GST标签后，纯化的rAahII（重组AahII）毒素，相对于天然AahII在其N端含有两个额外的氨基酸，在瑞士小鼠脑室内注射后具有高毒性。10 ng（或1.33 pmol）的LD50（半数致死剂量）值，接近天然毒素的LD50（3 ng），表明小麦胚体外翻译系统产生了正确折叠且具有生物活性的rAahII。此外，NbAahII10（澳大利亚杀人蝎纳米抗体10），一种针对天然AahII毒素产生的骆驼单域抗体片段，识别重组毒素上其同源的构象表位，并在注射到小鼠体内时中和纯化的rAahII的毒性。
