title: One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer.
abstract: We report an optimized method to purify and reconstitute histone octamer, which utilizes high expression of histones in inclusion bodies but eliminates the time consuming steps of individual histone purification. In the newly modified protocol, Xenopus laevis H2A, H2B, H3, and H4 are expressed individually into inclusion bodies of bacteria, which are subsequently mixed together and denatured in 8M guanidine hydrochloride. Histones are refolded and reconstituted into soluble octamer by dialysis against 2M NaCl, and metal-affinity purified through an N-terminal polyhistidine-tag added on the H2A. After cleavage of the polyhistidine-tag, histone octamer is further purified by size exclusion chromatography. We show that the nucleosomes reconstituted using the purified histone octamer above are fully functional. They serve as effective substrates for the histone methyltransferases DOT1L and MLL1. Small angle X-ray scattering further confirms that the reconstituted nucleosomes have correct structural integration of histone octamer and DNA as observed in the X-ray crystal structure. Our new protocol enables rapid reconstitution of histone octamer with an optimal yield. We expect this simplified approach to facilitate research using recombinant nucleosomes in vitro.
authors: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address: lyoungta@med.umich.edu.
authors: Young-Tae
authors: YT
authors: Lee
authors: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors: Garrett
authors: G
authors: Gibbons
authors: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors: Shirley Y
authors: SY
authors: Lee
authors: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors: Zaneta
authors: Z
authors: Nikolovska-Coleska
authors: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109, USA; Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA. Electronic address: yalid@med.umich.edu.
authors: Yali
authors: Y
authors: Dou
mesh_terms: D029845:Amphibian Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D019791:Guanidine / Q000737:chemistry; D011495:Histone-Lysine N-Methyltransferase / Q000737:chemistry; D006657:Histones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry*; D008780:Methyltransferases / Q000737:chemistry; D008958:Models, Molecular; D051788:Myeloid-Lymphoid Leukemia Protein / Q000737:chemistry; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D055503:Protein Multimerization; D058849:Protein Refolding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D029845:Amphibian Proteins; D006657:Histones; C497422:KMT2A protein, human; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; D051788:Myeloid-Lymphoid Leukemia Protein; D012965:Sodium Chloride; C473477:DOT1L protein, human; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D019791:Guanidine
keywords: Histone methyltransferase; Histone octamer; Nucleosome; Refolding; Small angle X-ray scattering