TRANSCRIPTION FACTOR-GREEN FLUORESCENT PROTEIN CHIMERIC FUSION PROTEINS AND THEIR USE IN STUDIES OF DNA AFFINITY-CHROMATOGRAPHY

A new plasmid, pJ22, was produced by introducing the enhanced green fl uorescent protein (GFP) coding sequence into the pET28 plasmid while r etaining much of the multiple cloning site. This new plasmid was then used to produce a chimeric fusion protein containing the DNA-binding r egion of the rat liver CAAT enhancer binding protein (C/EBP) fused to the CODH-terminus of GFP. This new GFP-C/EBP fusion protein also conta ins (His)(6) to facilitate purification by Ni2+-agarose and several ot her useful features. The plasmid and protein were developed to allow u s to more rapidly investigate the DNA-Sepharose affinity chromatograph y of transcription factors. The GFP-C/EBP protein is virtually identic al in its DNA-binding properties to a well-characterized, bacterially expressed protein called C/EBP 62 which has been shown to mimic rat wi ld-type C/EBP DNA-binding. GFP-C/EBP also binds to DNA-Sepharose which contains the CAAT element and is eluted by a salt gradient. Salt-depe ndent elution was highly temperature-dependent over the range of 4-19 degrees C. Since temperature-dependent DNA-binding has also been repor ted for other DNA-binding proteins, this may also occur with other tra nscription factors. DNA-affinity chromatography gave higher purity tha n that obtained by Ni2+-agarose chromatography and chromatography on t he same DNA-Sepharose column at two different temperatures resulted in the greatest purification, to near homogeneity. This temperature-depe ndent affinity chromatography provides an important new approach to tr anscription factor purification. (C) 1998 Elsevier Science B.V.