Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli

Microbial Cell Factories

Research

Nicolas Gilles¹  Vânia O. Fernandes²  Luís T. Gama²  Carlos M. G. A. Fontes³  Luís M. A. Ferreira³  Ana Filipa Sequeira³  Catarina I. P. I. Guerreiro⁴  Marilyne Blémont⁵  Renaud Vincentelli⁵  Laurie Ramond⁵  Hervé Darbon⁵  Fanny Peysson⁵  Jeremy Turchetto⁵  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;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;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;    Gene design;    Recombinant expression;    Periplasm;    Disulphide-rich peptides;    Fusion protein;    Escherichia coli (E. coli);    High-throughput expression;   

DOI  :  10.1186/s12934-016-0618-0

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

来源: Springer

【 摘 要 】

BackgroundAnimal 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.ResultsThe 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© The Author(s) 2017