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

PLoS One. 2013 May 7;8(5):e63865. doi: 10.1371/journal.pone.0063865. Print 2013.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Buffers
  • Disulfides / metabolism*
  • Escherichia coli / metabolism*
  • Genetic Vectors / genetics
  • Hydrogen-Ion Concentration
  • Magnetic Resonance Spectroscopy
  • Peptides / chemistry*
  • Peptides / metabolism*
  • Periplasm / metabolism*
  • Recombinant Proteins / biosynthesis*
  • Structure-Activity Relationship
  • Temperature


  • Buffers
  • Disulfides
  • Peptides
  • Recombinant Proteins

Grant support

Australian National Health and Medical Research Council (APP1044414) and Australian Research Council (FT100100027). NHMRC Project Grants APP1012338 and APP1034958 and Research Fellowship APP1044414 to GFK, as well as ARC Discovery Grants DP1095728 and DP110103129 and ARC Linkage Grant LP110100509. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.