High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery

Abstract

BackgroundAnimal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics.ResultsIn the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active.ConclusionsOverall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0617-1) contains supplementary material, which is available to authorized users.

Snake Venom Peptides: Tools of Biodiscovery

“…Apart from structural studies, different groups are working to generate large libraries of recombinant peptides by high-throughput expression of venom peptides in E. coli for drug discovery investigations [ 202 , 203 ]. Different metabolomics approaches are also used to analyze low-molecular-weight components of animal venoms [ 204 ].…”

Functional carbohydrate binding modules identified in evolved dits from siphophages infecting various Gram‐positive bacteria

“…For purification, plates were thawed for 10 min at 37°C in a water‐bath followed by 15 min shaking at 20°C in a Microtron shaking incubator. Protein purification was then performed as previously described (Saez and Vincentelli, , Turchetto et al, ).…”

Venomics-Accelerated Cone Snail Venom Peptide Discovery

“…To take advantage of the thousands of validated transcriptomic sequences available, a streamlined data-mining, production, and characterization pipeline is required that integrates high-throughput structural and pharmacological characterization with accelerated peptide production systems. Recently, a promising high-throughput recombinant expression of di-sulfide-reticulated venom peptides that can generate large libraries as an alternative to chemical synthesis was reported [ 69 ] that may start to fill this growing gap in the pipeline.…”