title: Expression of functional scorpion neurotoxin Lqq-V in E.coli.
abstract: We report the results on the expression in Escherichia coli of a functional neurotoxin LqqV from the scorpion Leiurus quinquestriatus quinquestriatus. The gene for LqqV was synthesized using recursive PCR and expressed as a poly-histidine-tagged fusion protein in thioredoxin mutant E. coli strain [AD494(DE3)pLysS], thus permitting disulfide-bond formation. When cultured at 37 degrees C, about 50% of the expressed protein is contained as a monomer in the soluble fraction of the E. coli extract. The fusion protein from the soluble fraction was purified and the His-tag was cleaved by thrombin, resulting in a yield of about 1.5 mg/liter. The globular structure of the purified protein was confirmed by NMR and CD spectroscopy. Patch-clamp measurements using native sodium channels in guinea pig ventricular myocytes reveal (1) a slowing of inactivation and (2) a decrease in peak current upon application of toxin, thus confirming the alpha-toxin activity of the purified recombinant protein.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, CH19-B31, 1530-3rd Ave. South, Birmingham, AL 35294-2041, USA.
authors.forename: Sami
authors.initials: S
authors.lastname: Banerjee
authors.forename: Ernest V
authors.initials: EV
authors.lastname: Curto
authors.forename: Matthew
authors.initials: M
authors.lastname: Beckman
authors.forename: George B
authors.initials: GB
authors.lastname: Brown
authors.forename: Juming
authors.initials: J
authors.lastname: Zhong
authors.forename: N Rama
authors.initials: NR
authors.lastname: Krishna
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D006168:Guinea Pigs; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D012604:Scorpion Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D012605:Scorpions
chemical_list: C538727:Lqq-V protein, Leiurus quinquestriatus; D009498:Neurotoxins; D012604:Scorpion Venoms