We report an expression strategy that uses chaperone-mediated folding in the periplasm to produce correctly-folded Bj-xtrIT β-scorpion toxin in E. coli. This represents the first reported instance of a disulfide-linked toxin purified directly from a bacterial expression system without the use of a fusion partner, or requiring solubilization of the expressed toxin from inclusion bodies, followed by refolding in vitro. Furthermore it yields a single conformation with the native disulfide connectivity. This expression strategy represents a time- and cost-effective solution that will enable routine toxin production and mutagenesis to aid in the investigation of channel structure–function relationship