Transcription factor-green fluorescent protein chimeric fusion proteins and their use in studies of DNA affinity...

A new plasmid, pJ22, was produced by introducing the enhanced green fluorescent protein (GFP) coding sequence into the pET28 plasmid while retaining much of the multiple cloning site. This new plasmid was then used to produce a chimeric fusion protein containing the DNA-binding region of the rat liver CAAT enhancer binding protein (C/EBP) fused to the COOH-terminus of GFP. This new GFP-C/EBP fusion protein also contains (His)6 to facilitate purification by Ni(2+)-agarose and several other useful features. The plasmid and protein were developed to allow us to more rapidly investigate the DNA-Sepharose affinity chromatography of transcription factors. The GFP-C/EBP protein is virtually identical in its DNA-binding properties to a well-characterized, bacterially expressed protein called C/EBP 62 which has been shown to mimic rat wild-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-dependent elution was highly temperature-dependent over the range of 4-19 degrees C. Since temperature-dependent DNA-binding has also been reported for other DNA-binding proteins, this may also occur with other transcription factors. DNA-affinity chromatography gave higher purity than that obtained by Ni(2+)-agarose chromatography and chromatography on the same DNA-Sepharose column at two different temperatures resulted in the greatest purification, to near homogeneity. This temperature-dependent affinity chromatography provides an important new approach to transcription factor purification.