High-Throughput Production of Oxidized Animal Toxins in Escherichia coli
| Autor: | Yoan Duhoo, Hervé Darbon, Renaud Vincentelli, Ana Filipa Sequeira, Joana L. A. Brás, Carlos M. G. A. Fontes, Natalie J. Saez, Laurie Ramond, Fanny Peysson, Nicolas Gilles, Jeremy Turchetto |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
chemistry.chemical_classification 0303 health sciences Drug discovery Chemistry Venom Peptide Periplasmic space Computational biology medicine.disease_cause 01 natural sciences law.invention 03 medical and health sciences Subcloning law 010608 biotechnology Protein biosynthesis medicine Recombinant DNA Escherichia coli 030304 developmental biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781493996230 |
| DOI: | 10.1007/978-1-4939-9624-7_7 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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