EXPRESSION, PURIFICATION, AND FUNCTIONAL-CHARACTERIZATION OF THE 2 ZINC-FINGER DOMAIN OF THE HUMAN GATA-1

The DNA-binding domain of the erythroid transcription factor GATA-1 co nsists of two closely related, but distinct zinc-fingers which are hig hly conserved among the members of the growing family of GATA-like fac tors. The DNA-binding domain of the human GATA-1 (F1F2) was expressed as a histidine-tagged fusion protein in Escherichia coli. The denatura ted protein was purified by Ni2+ chelate affinity chromatography and r enaturated irt situ. The active recombinant protein was purified by DN A affinity chromatography. F,F, displayed GATA-1 specific binding acti vity toward its DNA recognition sequences within the hypersensitive si te 3 of the human locus control region and the human gamma-globin prom oter. In contrast to GATA-1 protein purified from K562 nuclei, the rec ombinant F1F2 bound also the CCAAT-box region of the human gamma-globi n promoter. (C) 1994 Academic Press, Inc.