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 Ni(2+)-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 gamma-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F1F2 bound also the CCAAT-box region of the human gamma-globin promoter.

Expression, purification, and functional characterization of the two zinc-finger domain of the human GATA-1.

CALLIGARIS, RAFFAELLA;
1994

Abstract

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 Ni(2+)-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 gamma-globin promoter. In contrast to GATA-1 protein purified from K562 nuclei, the recombinant F1F2 bound also the CCAAT-box region of the human gamma-globin promoter.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11368/2769609
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