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High-Throughput Production of Oxidized Animal Toxins in Escherichia coli

Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

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.

Key words

  • Codon optimization
  • Gene synthesis
  • PCR assembly
  • High-Throughput
  • Codon optimization
  • PCR
  • Protein production
  • DsbC
  • Cysteine-rich proteins
  • Venom peptides
  • Toxins
  • Recombinant expression
  • Disulfide bonds

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  • DOI: 10.1007/978-1-4939-9624-7_7
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References

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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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Correspondence to Renaud Vincentelli .

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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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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9623-0

  • Online ISBN: 978-1-4939-9624-7

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