One-pot refolding of core histones from bacterial inclusion bodies allows rapid reconstitution of histone octamer

• Published in: Protein expression and purification 2015-06, Vol.110, p.89-94
• Main Authors: Lee, Young-Tae, Gibbons, Garrett, Lee, Shirley Y., Nikolovska-Coleska, Zaneta, Dou, Yali
• Format: Article
• Language: English
• Subjects: Amphibian Proteins - chemistry; Amphibian Proteins - genetics; Amphibian Proteins - isolation & purification; Animals; Cell Line; Cloning, Molecular; Crystallography, X-Ray; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Guanidine - chemistry; Histone methyltransferase; Histone octamer; Histone-Lysine N-Methyltransferase - chemistry; Histones - chemistry; Histones - genetics; Histones - isolation & purification; Humans; Inclusion Bodies - chemistry; Methyltransferases - chemistry; Models, Molecular; Myeloid-Lymphoid Leukemia Protein - chemistry; Nucleosome; Nucleosomes - chemistry; Nucleosomes - metabolism; Plasmids - chemistry; Plasmids - metabolism; Protein Multimerization; Protein Refolding; Protein Structure, Secondary; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - isolation & purification; Refolding; Small angle X-ray scattering; Sodium Chloride - chemistry; Solubility; Xenopus laevis - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.02.007

•We report a rapid method to reconstitute histone octamer by one-pot refolding.•The protocol eliminates time-consuming steps of individual histone purification.•Nucleosomes reconstituted from the purified octamer are fully functional.•Structural integrity of nucleosomes was confirmed by small angle X-ray scattering.•This protocol is expected to facilitate research on histone modifying enzymes. 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.