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. 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.