Expression,purification and functional characterization of the two zinc-fingers domain of the human GATA1

The DNA-binding domain of the erythroid transcription factor GATA-1 consists of two closely related, but distinct zinc-fingers which are highly conserved among the members of the growing family of GATA-like factors. The DNA-binding domain of the human GATA-1 (F₁F₂) was expressed as a histidine-tagged fusion protein in Escherichia coli. The denaturated protein was purified by Ni²⁺-chelate affinity chromatography and renaturated in situ. The active recombinant protein was purified by DNA affinity chromatography. F₁F₂ displayed GATA-1 specific binding activity toward its DNA recognition sequences within the hypersensitive site 3 of the human locus control region and the human γ-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F₁F₂ bound also the CCAAT-box region of the human γ-globin promoter.