High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery
Identifieur interne : 000265 ( Pmc/Checkpoint ); précédent : 000264; suivant : 000266High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery
Auteurs : Jeremy Turchetto [France] ; Ana Filipa Sequeira [Portugal] ; Laurie Ramond [France] ; Fanny Peysson [France] ; Joana L. A. Brás [Portugal] ; Natalie J. Saez [France, Australie] ; Yoan Duhoo [France] ; Marilyne Blémont [France] ; Catarina I. P. D. Guerreiro [Portugal] ; Loic Quinton [Belgique] ; Edwin De Pauw [Belgique] ; Nicolas Gilles [France] ; Hervé Darbon [France] ; Carlos M. G. A. Fontes [Portugal] ; Renaud Vincentelli [France]Source :
- Microbial Cell Factories [ 1475-2859 ] ; 2017.
Abstract
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.
In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in
Overall this study reveals that high-throughput expression of animal venom peptides in
The online version of this article (doi:10.1186/s12934-016-0617-1) contains supplementary material, which is available to authorized users.
Url:
DOI: 10.1186/s12934-016-0617-1
PubMed: 28095880
PubMed Central: 5242012
Affiliations:
- Australie, Belgique, France, Portugal
- Provence-Alpes-Côte d'Azur, Province de Liège
- Liège, Marseille
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PMC:5242012Le document en format XML
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Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Sequeira, Ana Filipa" sort="Sequeira, Ana Filipa" uniqKey="Sequeira A" first="Ana Filipa" last="Sequeira">Ana Filipa Sequeira</name><affiliation wicri:level="1"><nlm:aff id="Aff2">CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon</wicri:regionArea><wicri:noRegion>1300-477 Lisbon</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="Aff3">NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon</wicri:regionArea><wicri:noRegion>1649-038 Lisbon</wicri:noRegion></affiliation></author><author><name sortKey="Ramond, Laurie" sort="Ramond, Laurie" uniqKey="Ramond L" first="Laurie" last="Ramond">Laurie Ramond</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Peysson, Fanny" sort="Peysson, Fanny" uniqKey="Peysson F" first="Fanny" last="Peysson">Fanny Peysson</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Bras, Joana L A" sort="Bras, Joana L A" uniqKey="Bras J" first="Joana L. A." last="Brás">Joana L. A. Brás</name><affiliation wicri:level="1"><nlm:aff id="Aff3">NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon</wicri:regionArea><wicri:noRegion>1649-038 Lisbon</wicri:noRegion></affiliation></author><author><name sortKey="Saez, Natalie J" sort="Saez, Natalie J" uniqKey="Saez N" first="Natalie J." last="Saez">Natalie J. Saez</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation><affiliation wicri:level="1"><nlm:aff id="Aff4">Institute for Molecular Bioscience, The University of Queensland, St Lucia, 4072 Australia</nlm:aff><country xml:lang="fr">Australie</country><wicri:regionArea>Institute for Molecular Bioscience, The University of Queensland, St Lucia</wicri:regionArea><wicri:noRegion>St Lucia</wicri:noRegion></affiliation></author><author><name sortKey="Duhoo, Yoan" sort="Duhoo, Yoan" uniqKey="Duhoo Y" first="Yoan" last="Duhoo">Yoan Duhoo</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Blemont, Marilyne" sort="Blemont, Marilyne" uniqKey="Blemont M" first="Marilyne" last="Blémont">Marilyne Blémont</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Guerreiro, Catarina I P D" sort="Guerreiro, Catarina I P D" uniqKey="Guerreiro C" first="Catarina I. P. D." last="Guerreiro">Catarina I. P. D. Guerreiro</name><affiliation wicri:level="1"><nlm:aff id="Aff3">NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon</wicri:regionArea><wicri:noRegion>1649-038 Lisbon</wicri:noRegion></affiliation></author><author><name sortKey="Quinton, Loic" sort="Quinton, Loic" uniqKey="Quinton L" first="Loic" last="Quinton">Loic Quinton</name><affiliation wicri:level="3"><nlm:aff id="Aff5">Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000 Liège, Belgium</nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000 Liège</wicri:regionArea><placeName><region type="province" nuts="2">Province de Liège</region><settlement type="city">Liège</settlement></placeName></affiliation></author><author><name sortKey="De Pauw, Edwin" sort="De Pauw, Edwin" uniqKey="De Pauw E" first="Edwin" last="De Pauw">Edwin De Pauw</name><affiliation wicri:level="3"><nlm:aff id="Aff5">Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000 Liège, Belgium</nlm:aff><country xml:lang="fr">Belgique</country><wicri:regionArea>Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000 Liège</wicri:regionArea><placeName><region type="province" nuts="2">Province de Liège</region><settlement type="city">Liège</settlement></placeName></affiliation></author><author><name sortKey="Gilles, Nicolas" sort="Gilles, Nicolas" uniqKey="Gilles N" first="Nicolas" last="Gilles">Nicolas Gilles</name><affiliation wicri:level="1"><nlm:aff id="Aff6">CEA/DRF/iBiTecS, Service d’Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette, France</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>CEA/DRF/iBiTecS, Service d’Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette</wicri:regionArea><wicri:noRegion>91191 Gif-sur-Yvette</wicri:noRegion><wicri:noRegion>91191 Gif-sur-Yvette</wicri:noRegion></affiliation></author><author><name sortKey="Darbon, Herve" sort="Darbon, Herve" uniqKey="Darbon H" first="Hervé" last="Darbon">Hervé Darbon</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author><author><name sortKey="Fontes, Carlos M G A" sort="Fontes, Carlos M G A" uniqKey="Fontes C" first="Carlos M. G. A." last="Fontes">Carlos M. G. A. Fontes</name><affiliation wicri:level="1"><nlm:aff id="Aff2">CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon</wicri:regionArea><wicri:noRegion>1300-477 Lisbon</wicri:noRegion></affiliation><affiliation wicri:level="1"><nlm:aff id="Aff3">NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon, Portugal</nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea>NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon</wicri:regionArea><wicri:noRegion>1649-038 Lisbon</wicri:noRegion></affiliation></author><author><name sortKey="Vincentelli, Renaud" sort="Vincentelli, Renaud" uniqKey="Vincentelli R" first="Renaud" last="Vincentelli">Renaud Vincentelli</name><affiliation wicri:level="3"><nlm:aff id="Aff1">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</nlm:aff><country xml:lang="fr">France</country><wicri:regionArea>Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille</wicri:regionArea><placeName><region type="region">Provence-Alpes-Côte d'Azur</region><region type="old region">Provence-Alpes-Côte d'Azur</region><settlement type="city">Marseille</settlement></placeName></affiliation></author></analytic><series><title level="j">Microbial Cell Factories</title><idno type="eISSN">1475-2859</idno><imprint><date when="2017">2017</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><sec><title>Background</title><p>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.</p></sec><sec><title>Results</title><p>In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in <italic>Escherichia coli</italic> 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 <italic>E. coli</italic> 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.</p></sec><sec><title>Conclusions</title><p>Overall this study reveals that high-throughput expression of animal venom peptides in <italic>E. coli</italic> can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/s12934-016-0617-1) contains supplementary material, which is available to authorized users.</p></sec></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Escoubas, P" uniqKey="Escoubas P">P Escoubas</name></author><author><name sortKey="King, Gf" uniqKey="King G">GF King</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Calvete, Jj" uniqKey="Calvete J">JJ 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Microb Cell Fact</journal-id><journal-id journal-id-type="iso-abbrev">Microb. Cell Fact</journal-id><journal-title-group><journal-title>Microbial Cell Factories</journal-title></journal-title-group><issn pub-type="epub">1475-2859</issn><publisher><publisher-name>BioMed Central</publisher-name><publisher-loc>London</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">28095880</article-id><article-id pub-id-type="pmc">5242012</article-id><article-id pub-id-type="publisher-id">617</article-id><article-id pub-id-type="doi">10.1186/s12934-016-0617-1</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research</subject></subj-group></article-categories><title-group><article-title>High-throughput expression of animal venom toxins in <italic>Escherichia coli</italic> to generate a large library of oxidized disulphide-reticulated peptides for drug discovery</article-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Turchetto</surname><given-names>Jeremy</given-names></name><address><email>turchetto.jeremy@netcourrier.com</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Sequeira</surname><given-names>Ana Filipa</given-names></name><address><email>filipa.sequeira@nzytech.com</email></address><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Ramond</surname><given-names>Laurie</given-names></name><address><email>laurie.ramond@afmb.univ-mrs.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Peysson</surname><given-names>Fanny</given-names></name><address><email>fanny.peysson@orange.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Brás</surname><given-names>Joana L. A.</given-names></name><address><email>joana.bras@nzytech.com</email></address><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Saez</surname><given-names>Natalie J.</given-names></name><address><email>n.saez@imb.uq.edu.au</email></address><xref ref-type="aff" rid="Aff1">1</xref><xref ref-type="aff" rid="Aff4">4</xref></contrib><contrib contrib-type="author"><name><surname>Duhoo</surname><given-names>Yoan</given-names></name><address><email>yoan.duhoo@afmb.univ-mrs.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Blémont</surname><given-names>Marilyne</given-names></name><address><email>Marilyne.ROYANNEZ-BLEMONT@innate-pharma.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Guerreiro</surname><given-names>Catarina I. P. D.</given-names></name><address><email>catarina.guerreiro@nzytech.com</email></address><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author"><name><surname>Quinton</surname><given-names>Loic</given-names></name><address><email>loic.quinton@ulg.ac.be</email></address><xref ref-type="aff" rid="Aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>De Pauw</surname><given-names>Edwin</given-names></name><address><email>e.depauw@ulg.ac.be</email></address><xref ref-type="aff" rid="Aff5">5</xref></contrib><contrib contrib-type="author"><name><surname>Gilles</surname><given-names>Nicolas</given-names></name><address><email>Nicolas.GILLES@cea.fr</email></address><xref ref-type="aff" rid="Aff6">6</xref></contrib><contrib contrib-type="author"><name><surname>Darbon</surname><given-names>Hervé</given-names></name><address><email>Herve.Darbon@afmb.univ-mrs.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Fontes</surname><given-names>Carlos M. G. A.</given-names></name><address><email>carlos.fontes@nzytech.com</email></address><xref ref-type="aff" rid="Aff2">2</xref><xref ref-type="aff" rid="Aff3">3</xref></contrib><contrib contrib-type="author" corresp="yes"><contrib-id contrib-id-type="orcid">http://orcid.org/0000-0001-9667-0196</contrib-id><name><surname>Vincentelli</surname><given-names>Renaud</given-names></name><address><phone>+33 491825583</phone><email>renaud.vincentelli@afmb.univ-mrs.fr</email></address><xref ref-type="aff" rid="Aff1">1</xref></contrib><aff id="Aff1"><label>1</label>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</aff><aff id="Aff2"><label>2</label>CIISA-Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisbon, Portugal</aff><aff id="Aff3"><label>3</label>NZYtech Genes & Enzymes, Campus do Lumiar, Estrada do paço do Lumiar, 1649-038 Lisbon, Portugal</aff><aff id="Aff4"><label>4</label>Institute for Molecular Bioscience, The University of Queensland, St Lucia, 4072 Australia</aff><aff id="Aff5"><label>5</label>Mass Spectrometry Laboratory, B6c University of Liège, MolSys-Quartier Agora, Allée du six Aout 11, 4000 Liège, Belgium</aff><aff id="Aff6"><label>6</label>CEA/DRF/iBiTecS, Service d’Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette, France</aff></contrib-group><pub-date pub-type="epub"><day>17</day><month>1</month><year>2017</year></pub-date><pub-date pub-type="pmc-release"><day>17</day><month>1</month><year>2017</year></pub-date><pub-date pub-type="collection"><year>2017</year></pub-date><volume>16</volume><elocation-id>6</elocation-id><history><date date-type="received"><day>2</day><month>8</month><year>2016</year></date><date date-type="accepted"><day>16</day><month>12</month><year>2016</year></date></history><permissions><copyright-statement>© The Author(s) 2017</copyright-statement><license license-type="OpenAccess"><license-p><bold>Open Access</bold>This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/4.0/">http://creativecommons.org/licenses/by/4.0/</ext-link>), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (<ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/publicdomain/zero/1.0/">http://creativecommons.org/publicdomain/zero/1.0/</ext-link>) applies to the data made available in this article, unless otherwise stated.</license-p></license></permissions><abstract id="Abs1"><sec><title>Background</title><p>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.</p></sec><sec><title>Results</title><p>In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in <italic>Escherichia coli</italic> 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 <italic>E. coli</italic> 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.</p></sec><sec><title>Conclusions</title><p>Overall this study reveals that high-throughput expression of animal venom peptides in <italic>E. coli</italic> can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.</p></sec><sec><title>Electronic supplementary material</title><p>The online version of this article (doi:10.1186/s12934-016-0617-1) contains supplementary material, which is available to authorized users.</p></sec></abstract><kwd-group xml:lang="en"><title>Keywords</title><kwd>Venom peptides</kwd><kwd>Disulphide bonds</kwd><kwd>Periplasm</kwd><kwd><italic>Escherichia coli</italic> (<italic>E. coli</italic>)</kwd><kwd>Drug discovery library</kwd><kwd>High-throughput production</kwd></kwd-group><funding-group><award-group><funding-source><institution>VENOMICS FP7 grant</institution></funding-source><award-id>278346</award-id><principal-award-recipient><name><surname>Turchetto</surname><given-names>Jeremy</given-names></name></principal-award-recipient></award-group><award-group><funding-source><institution>VENOMICS</institution></funding-source><award-id>278346</award-id><award-id>278346</award-id><award-id>278346</award-id><award-id>278346</award-id><principal-award-recipient><name><surname>Ramond</surname><given-names>Laurie</given-names></name><name><surname>Peysson</surname><given-names>Fanny</given-names></name><name><surname>Saez</surname><given-names>Natalie J.</given-names></name><name><surname>Blémont</surname><given-names>Marilyne</given-names></name></principal-award-recipient></award-group><award-group><funding-source><institution-wrap><institution-id institution-id-type="FundRef">http://dx.doi.org/10.13039/501100001871</institution-id><institution>Fundação para a Ciência e a Tecnologia</institution></institution-wrap></funding-source><award-id>SFRH/BD/51602/2011</award-id><principal-award-recipient><name><surname>Sequeira</surname><given-names>Ana Filipa</given-names></name></principal-award-recipient></award-group></funding-group><custom-meta-group><custom-meta><meta-name>issue-copyright-statement</meta-name><meta-value>© The Author(s) 2017</meta-value></custom-meta></custom-meta-group></article-meta></front></pmc><affiliations><list><country><li>Australie</li><li>Belgique</li><li>France</li><li>Portugal</li></country><region><li>Provence-Alpes-Côte d'Azur</li><li>Province de Liège</li></region><settlement><li>Liège</li><li>Marseille</li></settlement></list><tree><country name="France"><region name="Provence-Alpes-Côte d'Azur"><name sortKey="Turchetto, Jeremy" sort="Turchetto, Jeremy" uniqKey="Turchetto J" first="Jeremy" last="Turchetto">Jeremy Turchetto</name></region><name sortKey="Blemont, Marilyne" sort="Blemont, Marilyne" uniqKey="Blemont M" first="Marilyne" last="Blémont">Marilyne Blémont</name><name sortKey="Darbon, Herve" sort="Darbon, Herve" uniqKey="Darbon H" first="Hervé" last="Darbon">Hervé Darbon</name><name sortKey="Duhoo, Yoan" sort="Duhoo, Yoan" uniqKey="Duhoo Y" first="Yoan" last="Duhoo">Yoan Duhoo</name><name sortKey="Gilles, Nicolas" sort="Gilles, Nicolas" uniqKey="Gilles N" first="Nicolas" last="Gilles">Nicolas Gilles</name><name sortKey="Peysson, Fanny" sort="Peysson, Fanny" uniqKey="Peysson F" first="Fanny" last="Peysson">Fanny Peysson</name><name sortKey="Ramond, Laurie" sort="Ramond, Laurie" uniqKey="Ramond L" first="Laurie" last="Ramond">Laurie Ramond</name><name sortKey="Saez, Natalie J" sort="Saez, Natalie J" uniqKey="Saez N" first="Natalie J." last="Saez">Natalie J. Saez</name><name sortKey="Vincentelli, Renaud" sort="Vincentelli, Renaud" uniqKey="Vincentelli R" first="Renaud" last="Vincentelli">Renaud Vincentelli</name></country><country name="Portugal"><noRegion><name sortKey="Sequeira, Ana Filipa" sort="Sequeira, Ana Filipa" uniqKey="Sequeira A" first="Ana Filipa" last="Sequeira">Ana Filipa Sequeira</name></noRegion><name sortKey="Bras, Joana L A" sort="Bras, Joana L A" uniqKey="Bras J" first="Joana L. A." last="Brás">Joana L. A. Brás</name><name sortKey="Fontes, Carlos M G A" sort="Fontes, Carlos M G A" uniqKey="Fontes C" first="Carlos M. G. A." last="Fontes">Carlos M. G. A. Fontes</name><name sortKey="Fontes, Carlos M G A" sort="Fontes, Carlos M G A" uniqKey="Fontes C" first="Carlos M. G. A." last="Fontes">Carlos M. G. A. Fontes</name><name sortKey="Guerreiro, Catarina I P D" sort="Guerreiro, Catarina I P D" uniqKey="Guerreiro C" first="Catarina I. P. D." last="Guerreiro">Catarina I. P. D. Guerreiro</name><name sortKey="Sequeira, Ana Filipa" sort="Sequeira, Ana Filipa" uniqKey="Sequeira A" first="Ana Filipa" last="Sequeira">Ana Filipa Sequeira</name></country><country name="Australie"><noRegion><name sortKey="Saez, Natalie J" sort="Saez, Natalie J" uniqKey="Saez N" first="Natalie J." last="Saez">Natalie J. Saez</name></noRegion></country><country name="Belgique"><region name="Province de Liège"><name sortKey="Quinton, Loic" sort="Quinton, Loic" uniqKey="Quinton L" first="Loic" last="Quinton">Loic Quinton</name></region><name sortKey="De Pauw, Edwin" sort="De Pauw, Edwin" uniqKey="De Pauw E" first="Edwin" last="De Pauw">Edwin De Pauw</name></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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