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

Julie K Klint,Niraj S Bende,Glenn F King,Eivind A B Undheim,Sebastian Senff,Natalie J Saez,Ho Yee Lau,Radha Seshadri,Lachlan D Rash,Mehdi Mobli,Eric Cascales

doi:10.1371/journal.pone.0063865

Abstract

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.

Highlights

Animal venoms are gaining increased attention as a source of novel bioactive peptides [1,2]
What do we do? When comparing the yield of fusion protein between cultures grown in Luria-Bertani medium (LB), MM, or the dual-media method, we find that the final yield of fusion protein remains essentially constant regardless of the choice of labelling method (Fig. 2, step 3)
Step 6 – How do I cleave my peptide from the fusion tag? What can you do? We focus here on Tobacco etch virus (TEV) protease cleavage since our vector encodes a TEV protease cleavage site immediately prior to the target peptide sequence

Summary

Introduction

Animal venoms are gaining increased attention as a source of novel bioactive peptides [1,2]. The recommended reaction conditions for TEV protease cleavage include 1 mM DTT or 0.5 mM TCEP as a reducing agent, or a redox pair (3.0 mM reduced GSH/0.3 mM oxidized GSH) for proteins containing disulfide bonds [71] These concentrations of GSH and oxidized GSH (GSSG) will yield a solution redox potential of –260 mV (calculated using the Nernst equation [72]). Using a syringe-driven solid-phase extraction (SPE) column offers a facile alternative for separation of the large hydrophobic components (TEV protease and MBP) from the target peptide, even if the latter is somewhat hydrophobic The disadvantage of this method is that one needs to first determine the amount and volume of organic solvent needed to elute the peptide, but not the fusion tags. The lowest protein concentration that could be reliably measured using the NanoDrop was 20 mM and the standard error was 5 mM

Assay Method Concentration

Findings

Materials and Methods