title: Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins.
abstract: 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.
authors.forename: Wael
authors.initials: W
authors.lastname: Gad
authors.affiliation: §Laboratoire des Venins et Molécules Thérapeutiques, Institut Pasteur de Tunis-Université Tunis El Manar, Tunisia.
authors.forename: Rahma
authors.initials: R
authors.lastname: Ben-Abderrazek
authors.forename: Khadija
authors.initials: K
authors.lastname: Wahni
authors.affiliation: ∥de Duve Institute, Université Catholique de Louvain, 1200 Brussels, Belgium.
authors.forename: Didier
authors.initials: D
authors.lastname: Vertommen
authors.forename: Serge
authors.initials: S
authors.lastname: Muyldermans
authors.forename: Balkiss
authors.initials: B
authors.lastname: Bouhaouala-Zahar
authors.forename: Joris
authors.initials: J
authors.lastname: Messens
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D005982:Glutathione Transferase / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D012604:Scorpion Venoms / Q000378:metabolism*; D012605:Scorpions / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D014908:Triticum / Q000378:metabolism*
chemical_list: D010455:Peptides; D012604:Scorpion Venoms; D015222:Sodium Channels; C053969:scorpion toxin II, Androctonus; D005982:Glutathione Transferase