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Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli
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...
Autores principales: | , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240416/ https://www.ncbi.nlm.nih.gov/pubmed/28093085 http://dx.doi.org/10.1186/s12934-016-0618-0 |
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author | Sequeira, Ana Filipa Turchetto, Jeremy Saez, Natalie J. Peysson, Fanny Ramond, Laurie Duhoo, Yoan Blémont, Marilyne Fernandes, Vânia O. Gama, Luís T. Ferreira, Luís M. A. Guerreiro, Catarina I. P. I. Gilles, Nicolas Darbon, Hervé Fontes, Carlos M. G. A. Vincentelli, Renaud |
author_facet | Sequeira, Ana Filipa Turchetto, Jeremy Saez, Natalie J. Peysson, Fanny Ramond, Laurie Duhoo, Yoan Blémont, Marilyne Fernandes, Vânia O. Gama, Luís T. Ferreira, Luís M. A. Guerreiro, Catarina I. P. I. Gilles, Nicolas Darbon, Hervé Fontes, Carlos M. G. A. Vincentelli, Renaud |
author_sort | Sequeira, Ana Filipa |
collection | PubMed |
description | 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0618-0) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5240416 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-52404162017-01-23 Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli Sequeira, Ana Filipa Turchetto, Jeremy Saez, Natalie J. Peysson, Fanny Ramond, Laurie Duhoo, Yoan Blémont, Marilyne Fernandes, Vânia O. Gama, Luís T. Ferreira, Luís M. A. Guerreiro, Catarina I. P. I. Gilles, Nicolas Darbon, Hervé Fontes, Carlos M. G. A. Vincentelli, Renaud Microb Cell Fact Research 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12934-016-0618-0) contains supplementary material, which is available to authorized users. BioMed Central 2017-01-17 /pmc/articles/PMC5240416/ /pubmed/28093085 http://dx.doi.org/10.1186/s12934-016-0618-0 Text en © The Author(s) 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), 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 (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Sequeira, Ana Filipa Turchetto, Jeremy Saez, Natalie J. Peysson, Fanny Ramond, Laurie Duhoo, Yoan Blémont, Marilyne Fernandes, Vânia O. Gama, Luís T. Ferreira, Luís M. A. Guerreiro, Catarina I. P. I. Gilles, Nicolas Darbon, Hervé Fontes, Carlos M. G. A. Vincentelli, Renaud Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title | Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title_full | Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title_fullStr | Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title_full_unstemmed | Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title_short | Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli |
title_sort | gene design, fusion technology and tev cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in escherichia coli |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240416/ https://www.ncbi.nlm.nih.gov/pubmed/28093085 http://dx.doi.org/10.1186/s12934-016-0618-0 |
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