Purification and characterization of the DNA-binding domain of BTEB, a GC box-binding transcription factor, expressed in Escherichia coli

Yasuo Kikuchi, Kazuhiro Sogawa, Nobuaki Watanabe, Akira Kobayashi, Yoshiaki Fujii-Kuriyama

    研究成果: Article査読

    6 被引用数 (Scopus)

    抄録

    BTEB is a GC-binding protein that regulates the transcription of genes with a single GC-box or tandemly repeated GC-boxes in the promoter. The DNA-binding domain of BTEB consists of three contiguous Cys2-His2 zinc finger motifs and short segments adjacent to their N- and C-terminal sides. The truncated BTEB (residues 120 to 244) containing the DNA-binding domain was expressed in Escherichia call and purified to homogeneity under denaturing conditions. DNA-binding activity of the BTEB was regenerated by refolding in the presence of Zn2+. The efficiency in regeneration was 70 ± 10%, and the dissociation constant (K(d)) of the DNA-complex was 4 ± 2 nM. Co2+ also regenerated the DNA-binding affinity of BTEB, albeit with less efficiency than Zn2+. Co-BTEB showed a slightly lower affinity to the specific DNA than Zn-BTEB. Refolding in the presence of Cd2+ resulted in an extremely low efficiency in regeneration of the DNA-binding activity. Zn-BTEB is in a monomer state at concentrations lower than 0.5 μM, and forms a dimer in the concentration range of about 10 to 200 μM.

    本文言語English
    ページ(範囲)309-313
    ページ数5
    ジャーナルJournal of biochemistry
    119
    2
    DOI
    出版ステータスPublished - 1996 2月

    ASJC Scopus subject areas

    • 生化学
    • 分子生物学

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