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

    Research output: Contribution to journalArticle

    5 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)309-313
    Number of pages5
    JournalJournal of biochemistry
    Volume119
    Issue number2
    DOIs
    Publication statusPublished - 1996 Feb

    Keywords

    • DNA-binding protein
    • GC-box
    • Homodimerization
    • Transcription factor
    • Zinc finger

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

    Fingerprint Dive into the research topics of 'Purification and characterization of the DNA-binding domain of BTEB, a GC box-binding transcription factor, expressed in Escherichia coli'. Together they form a unique fingerprint.

    Cite this