Abstract
Epigenetic mechanisms underlie the morphological transformations and shifts in virulence of eukaryotic pathogens. The targeting of epigenetics-driven cellular programs thus represents an Achilles’ heel of human parasites. Today, zinc-dependent histone deacetylases (HDACs) belong to the most explored epigenetic drug targets in eukaryotic parasites. Here, we describe an optimized protocol for the large-scale overproduction and purification of recombinant smHDAC8, an emerging epigenetic drug target in the multicellular human-pathogenic flatworm Schistosoma mansoni. The strategy employs the robustness of recombinant expression in Escherichia coli together with initial purification through a poly-histidine affinity tag that can be removed by the thrombin protease. This protocol is divided into two steps: (1) large-scale production of smHDAC8 in E. coli, and (2) purification of the target smHDAC8 protein through multiple purification steps.
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Acknowledgements
This work and the authors of this manuscript have been supported by funding from the European Union’s Seventh Framework Programme for research, technological development and demonstration under grant agreements nos. 241865 (SEtTReND) and 602080 (A-ParaDDisE). The authors are supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Université de Strasbourg and the Université de Lille 2, the French Infrastructure for Integrated Structural Biology (FRISBI; ANR-10-INSB-05-01), and by Instruct as part of the European Strategy Forum on Research Infrastructures (ESFRI).
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Marek, M., Shaik, T.B., Duclaud, S., Pierce, R.J., Romier, C. (2016). Large-Scale Overproduction and Purification of Recombinant Histone Deacetylase 8 (HDAC8) from the Human-Pathogenic Flatworm Schistosoma mansoni . In: Sarkar, S. (eds) Histone Deacetylases. Methods in Molecular Biology, vol 1436. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3667-0_8
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DOI: https://doi.org/10.1007/978-1-4939-3667-0_8
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3667-0
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