Expression, purification, and functional characterization of the two zinc-finger domain of the human GATA-1.
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.
Apezteguia
I
I
Interuniversitary Consortium for Biotechnology-National Laboratory, Trieste, Italy.
Calligaris
R
R
Bottardi
S
S
Santoro
C
C
D001483:Base Sequence; D002846:Chromatography, Affinity; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D050981:Erythroid-Specific DNA-Binding Factors; D004926:Escherichia coli / Q000378:metabolism; D050982:GATA1 Transcription Factor; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D009532:Nickel; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014157:Transcription Factors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D011994:Recombinant Proteins; D014157:Transcription Factors; D006639:Histidine; D009532:Nickel