Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli
Open Access
- 7 May 2013
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 8 (5), e63865
- https://doi.org/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.Keywords
This publication has 113 references indexed in Scilit:
- An overview of enzymatic reagents for the removal of affinity tagsProtein Expression and Purification, 2011
- Gene optimization mechanisms: A multi‐gene study reveals a high success rate of full‐length human proteins expressed in Escherichia coliProtein Science, 2010
- A Bivalent Tarantula Toxin Activates the Capsaicin Receptor, TRPV1, by Targeting the Outer Pore DomainCell, 2010
- The optimization of in vitro high-throughput chemical lysis of Escherichia coli. Application to ACP domain of the polyketide synthase ppsC from Mycobacterium tuberculosisJournal of Structural and Functional Genomics, 2010
- Practical protocols for production of very high yields of recombinant proteins using Escherichia coliProtein Science, 2009
- Integrated Oxidative Folding of Cysteine/Selenocysteine Containing Peptides: Improving Chemical Synthesis of ConotoxinsAngewandte Chemie-International Edition, 2009
- AChBP-targeted α-conotoxin correlates distinct binding orientations with nAChR subtype selectivityThe EMBO Journal, 2007
- The Strep-tag system for one-step purification and high-affinity detection or capturing of proteinsNature Protocols, 2007
- Secretory and extracellular production of recombinant proteins using Escherichia coliApplied Microbiology and Biotechnology, 2004
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976