title: Large-Scale Overproduction and Purification of Recombinant Histone Deacetylase 8 (HDAC8) from the Human-Pathogenic Flatworm Schistosoma mansoni.
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.
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404, Illkirch Cedex, France.
authors.forename: Martin
authors.initials: M
authors.lastname: Marek
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404, Illkirch Cedex, France.
authors.forename: Tajith B
authors.initials: TB
authors.lastname: Shaik
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404, Illkirch Cedex, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Duclaud
authors.affiliation: Center for Infection and Immunity of Lille (CIIL), INSERM U1019 - CNRS UMR 8204, Université de Lille, Institut Pasteur de Lille, 1 rue Professeur Calmette, 59019, Lille Cedex, France.
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Pierce
authors.affiliation: Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries, B.P. 10142, 67404, Illkirch Cedex, France. romier@igbmc.fr.
authors.forename: Christophe
authors.initials: C
authors.lastname: Romier
mesh_terms: D000818:Animals; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D030781:Organisms, Genetically Modified; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012550:Schistosoma mansoni / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D012097:Repressor Proteins; C409470:HDAC8 protein, human; D006655:Histone Deacetylases
keywords: Enzyme; Histone deacetylase; Purification; Recombinant expression; Schistosoma