title: High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.
abstract: BACKGROUND
  Animal 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.
  
  
  RESULTS
  In 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.
  
  
  CONCLUSIONS
  Overall 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.
authors.affiliation: 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.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Turchetto
authors.affiliation: 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.
authors.forename: Ana Filipa
authors.initials: AF
authors.lastname: Sequeira
authors.affiliation: 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.
authors.forename: Laurie
authors.initials: L
authors.lastname: Ramond
authors.affiliation: 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.
authors.forename: Fanny
authors.initials: F
authors.lastname: Peysson
authors.affiliation: NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038, Lisbon, Portugal.
authors.forename: Joana L A
authors.initials: JL
authors.lastname: Brás
authors.affiliation: 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, St Lucia, 4072, Australia.
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Saez
authors.affiliation: 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.
authors.forename: Yoan
authors.initials: Y
authors.lastname: Duhoo
authors.affiliation: 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.
authors.forename: Marilyne
authors.initials: M
authors.lastname: Blémont
authors.affiliation: NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038, Lisbon, Portugal.
authors.forename: Catarina I P D
authors.initials: CI
authors.lastname: Guerreiro
authors.affiliation: Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000, Liège, Belgium.
authors.forename: Loic
authors.initials: L
authors.lastname: Quinton
authors.affiliation: Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000, Liège, Belgium.
authors.forename: Edwin
authors.initials: E
authors.lastname: De Pauw
authors.affiliation: CEA/DRF/iBiTecS, Service d'Ingénierie Moléculaire des Protéines, 91191, Gif-sur-Yvette, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Gilles
authors.affiliation: 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.
authors.forename: Hervé
authors.initials: H
authors.lastname: Darbon
authors.affiliation: 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.
authors.forename: Carlos M G A
authors.initials: CM
authors.lastname: Fontes
authors.affiliation: 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. renaud.vincentelli@afmb.univ-mrs.fr.
authors.forename: Renaud
authors.identifier: http://orcid.org/0000-0001-9667-0196
authors.initials: R
authors.lastname: Vincentelli
mesh_terms: D000818:Animals; D004220:Disulfides / Q000737:chemistry; D055808:Drug Discovery / Q000379:methods; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D010084:Oxidation-Reduction; D019151:Peptide Library; D010455:Peptides / Q000235:genetics* / Q000302:isolation & purification / Q000627:therapeutic use; D019897:Periplasm / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000627:therapeutic use; D014688:Venoms / Q000737:chemistry / Q000235:genetics*
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D014688:Venoms; D010450:Endopeptidases; C099488:TEV protease
keywords: Disulphide bonds; Drug discovery library; Escherichia coli (E. coli); High-throughput production; Periplasm; Venom peptides