Temperature dependence of DNA affinity chromatography of transcription factors

Oligonucleotides bound by the CAAT enhancer binding protein (C/EBP), the la ctose repressor, and Gal4 were chemically coupled to cyanogen bromide-activ ated Sepharose and the temperature dependence of transcription factor chrom atography was characterized, Each transcription factor was applied to the a p appropriate column and eluted using a salt gradient at several temperatur es, Each transcription factor showed a unique behavior. As temperature was increases, less salt was required to elute C/EBP, more salt was required to elute lac repressor, while Gal4 showed a biphasic dependency with the amou nt of salt first decreasing between 4 and 19 degrees C and then increasing above 19 degrees C. This temperature dependence is not due to protein or DN A unfolding but rather is a property of complex formation. By loading a col umn, washing it at a permissive temperature, and then rapidly changing the column temperature, highly selective elution can be obtained. The thermodyn amics of this temperature effect are different for the binding of specific and nonspecific DNA sequences, making chromatography at different temperatu res a potentially important way of purifying transcription factors, (C) 200 0 Academic Press.