Comisso viaggiatore in Oriente

β-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Na v channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv 2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav 1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNa v 1 resistance to Dc1a, we identified two residues within the BgNav 1 domain II S1-S2 loop that when mutated to their PaNav 1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides. © 2014 Macmillan Publishers Limited. All rights reserved

Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds