Expression and purification of recombinant human histones

Yoshinori Tanaka, Maki Tawaramoto-Sasanuma, Shinichi Kawaguchi, Tsutomu Ohta, Kinya Yoda, Hitoshi Kurumizaka, Shigeyuki Yokoyama

    研究成果: Article

    117 引用 (Scopus)

    抜粋

    Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.

    元の言語English
    ページ(範囲)3-11
    ページ数9
    ジャーナルMethods
    33
    発行部数1
    DOI
    出版物ステータスPublished - 2004 5

      フィンガープリント

    ASJC Scopus subject areas

    • Molecular Biology

    これを引用

    Tanaka, Y., Tawaramoto-Sasanuma, M., Kawaguchi, S., Ohta, T., Yoda, K., Kurumizaka, H., & Yokoyama, S. (2004). Expression and purification of recombinant human histones. Methods, 33(1), 3-11. https://doi.org/10.1016/j.ymeth.2003.10.024