Abstract
High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.
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References
Robinson SD, Undheim EAB, Ueberheide B, King GF (2017) Venom peptides as therapeutics: advances, challenges and the future of venom-peptide discovery. Expert Rev Proteomics 14(10):931–939. https://doi.org/10.1080/14789450.2017.1377613
Sequeira AF, Turchetto J, Saez NJ, Peysson F, Ramond L, Duhoo Y, Blemont M, Fernandes VO, Gama LT, Ferreira LM, Guerreiro CI, Gilles N, Darbon H, Fontes CM, Vincentelli R (2017) Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli. Microb Cell Factories 16(1):4. https://doi.org/10.1186/s12934-016-0618-0
Turchetto J, Sequeira AF, Ramond L, Peysson F, Bras JL, Saez NJ, Duhoo Y, Blemont M, Guerreiro CI, Quinton L, De Pauw E, Gilles N, Darbon H, Fontes CM, Vincentelli R (2017) High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery. Microb Cell Factories 16(1):6. https://doi.org/10.1186/s12934-016-0617-1
Saez NJ, Nozach H, Blemont M, Vincentelli R (2014) High throughput quantitative expression screening and purification applied to recombinant disulfide-rich venom proteins produced in E. coli. J Vis Exp 89:e51464. https://doi.org/10.3791/51464
van den Berg S, Lofdahl PA, Hard T, Berglund H (2006) Improved solubility of TEV protease by directed evolution. J Biotechnol 121(3):291–298. https://doi.org/10.1016/j.jbiotec.2005.08.006
Acknowledgements
This work was supported by The VENOMICS European project grant N° 278346 through the Seventh Framework Program (FP7 HEALTH 2011-2015). NZYTech gratefully acknowledges PORTUGAL 2020—Programa Operacional Regional de Lisboa, Project 011199. AFMB was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01.
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Duhoo, Y. et al. (2019). High-Throughput Production of Oxidized Animal Toxins in Escherichia coli. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_7
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DOI: https://doi.org/10.1007/978-1-4939-9624-7_7
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