Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli

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.

Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target NaV1.7

“…A synthetic gene facilitating periplasmic expression (GeneArt, Invitrogen, Regensburg, Germany) encoding Hhn2b was used as previously described (Klint et al, 2013). Site-directed mutagenesis was performed by the quick-change polymerase chain reaction method using standard protocols with Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad, CA) (Qi and Scholthof, 2008).…”

“…Peptides were produced following an optimized protocol described previously (Klint et al, 2013). Peptide purity was assessed by analytical high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and all peptides used showed .95% purity.…”

Identification and Characterization of ProTx-III [μ-TRTX-Tp1a], a New Voltage-Gated Sodium Channel Inhibitor from Venom of the Tarantula Thrixopelma pruriens

“…Recombinant Tp1a was readily produced using a bacterial expression system as reported for other ICK peptides (Saez et al, 2011;Klint et al, 2013), although the recombinant version had a non-native N-terminal Gly residue, and the C terminus was not amidated as in the native toxin. In contrast, chemical synthesis of Tp1a allowed production of both the Positively and negatively charged residues are highlighted in blue and red, respectively.…”

“…A synthetic gene encoding Tp1a, with codons optimized for expression in Escherichia coli, was produced and cloned into the pLicC vector by GeneArt (Life Technologies). This vector contains a MalE signal sequence for periplasmic expression, a His 6 tag for nickel affinity purification, a maltose binding protein (MBP) tag to enhance solubility, and a tobacco etch virus (TEV) protease recognition site preceding the Tp1a gene (Klint et al, 2013). The expression plasmid was transformed in E. coli strain BL21(DE3) and cells cultured in Luria-Bertani medium at 37°C, 180 rpm.…”

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

“…Potential variables to be optimized include, but are not limited to, varying expression strains 1,2 , temperature 3,4 , media 2,3 , target variants 5 , fusion partners [6][7][8][9][10][11][12][13] , co-expression with chaperones 14,15 , cytoplasmic or periplasmic expression [16][17][18] , and purification buffer components 3 . By implementing high throughput approaches, many variables or many targets can be tested in parallel with a high level of efficiency, while limiting batch-to-batch variation.…”