title: Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.
abstract: BACKGROUND
  Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli.
  
  
  RESULTS
  The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal.
  
  
  CONCLUSIONS
  This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.
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: Jeremy
authors.initials: J
authors.lastname: Turchetto
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: Fanny
authors.initials: F
authors.lastname: Peysson
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: 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: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal.
authors.forename: Vânia O
authors.initials: VO
authors.lastname: Fernandes
authors.affiliation: CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477, Lisbon, Portugal.
authors.forename: Luís T
authors.initials: LT
authors.lastname: Gama
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: Luís M A
authors.initials: LM
authors.lastname: Ferreira
authors.affiliation: NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038, Lisbon, Portugal.
authors.forename: Catarina I P I
authors.initials: CI
authors.lastname: Guerreiro
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; D001709:Biotechnology / Q000379:methods; D003001:Cloning, Molecular; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D057166:High-Throughput Screening Assays; D010084:Oxidation-Reduction; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D019897:Periplasm / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility; D014688:Venoms / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D004220:Disulfides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D014688:Venoms; D010450:Endopeptidases; C099488:TEV protease
keywords: Disulphide-rich peptides; Escherichia coli (E. coli); Fusion protein; Gene design; High-throughput expression; Periplasm; Recombinant expression; Venom peptides