Functional Expression of a ß-Scorpion Toxin in the E.Coli Periplasm: A Tool for Exploring Sodium Channel/Ligand Interactions

Abstract

β-scorpion toxins are polypeptides of 60-76 residues that bind to site 4 of voltage-gated sodium channels and induce a hyper-polarising shift in the voltage-dependence of activation. The domain II voltage sensor is trapped in its outward, activated conformation through interactions with the toxin. β-scorpion toxins represent lead candidates for novel therapeutics and insecticides but their expression in bacterial systems is not straight-forward; the reducing cytoplasmic environment hinders formation of four disulphide bridges that stabilise the toxin structure. Therefore reports to-date of recombinant β-toxin production include an oxidative-refolding step which follows initial purification of the mis-folded peptide.We report an expression strategy that produces correctly-folded β-toxin in E.coli. FPLC and HPLC analysis of β-toxin purified to homogeneity indicted that the protein adopts a single conformation. Crystallization and structure determination to 2.5 A resolution confirmed that the β-toxin adopts the functionally-active form. Additionally, thermal denaturation studies using synchrotron-radiation circular dichroism (SRCD) – the first application of this sensitive CD technique to this toxin class – demonstrated the stability of this cross-linked toxin.The methodology applied to produce this toxin may facilitate industrial scale-up or lab-based production of wild-type and mutant toxins for ligand interaction studies.