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

Microbial Cell Factories

Research

Nicolas Gilles¹  Carlos M. G. A. Fontes²  Ana Filipa Sequeira²  Edwin De Pauw³  Loic Quinton³  Catarina I. P. D. Guerreiro⁴  Joana L. A. Brás⁴  Marilyne Blémont⁵  Jeremy Turchetto⁵  Renaud Vincentelli⁵  Laurie Ramond⁵  Hervé Darbon⁵  Fanny Peysson⁵  Yoan Duhoo⁵  Natalie J. Saez⁶ 

[1] CEA/DRF/iBiTecS, Service d’Ingénierie Moléculaire des Protéines, 91191, Gif-sur-Yvette, France;CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal;NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038, Lisbon, Portugal;Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000, Liège, Belgium;NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038, Lisbon, Portugal;Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France;Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France;Institute for Molecular Bioscience, The University of Queensland, 4072, St Lucia, Australia;

关键词: Venom peptides;    Disulphide bonds;    Periplasm;    Escherichia coli;    Drug discovery library;    High-throughput production;   

DOI  :  10.1186/s12934-016-0617-1

 received in 2016-08-02, accepted in 2016-12-16,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s) 2017