Expression of functional scorpion neurotoxin Lqq-V in E.coli.

Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, CH19-B31, 1530-3rd Ave. South, Birmingham, AL 35294-2041, USA.
Peptides (Impact Factor: 2.61). 02/2006; 27(1):49-54. DOI: 10.1016/j.peptides.2005.06.023
Source: PubMed

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.