Purification and characterization of the DNA-binding domain of BTEB, a GC box-binding transcription factor, expressed in Escherichia coli.
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 [Kobayashi et al. (1995) J. Biochem. 117, 91-95]. The truncated BTEB (residues 120 to 244) containing the DNA-binding domain was expressed in Escherichia coli 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 (Kd) 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 microM, and forms a dimer in the concentration range of about 10 to 200 microM.
Kikuchi
Y
Y
Department of Chemistry, Tohoku University, Sendai.
Sogawa
K
K
Watanabe
N
N
Kobayashi
A
A
Fujii-Kuriyama
Y
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016335:Zinc Fingers
D017931:DNA Primers; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D014157:Transcription Factors; C077017:Klf9 protein, rat