Temperature dependence of DNA affinity chromatography of transcription factors.
Oligonucleotides bound by the CAAT enhancer binding protein (C/EBP), the lactose repressor, and Gal4 were chemically coupled to cyanogen bromide-activated Sepharose and the temperature dependence of transcription factor chromatography was characterized. Each transcription factor was applied to the appropriate column and eluted using a salt gradient at several temperatures. 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 amount 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 DNA unfolding but rather is a property of complex formation. By loading a column, washing it at a permissive temperature, and then rapidly changing the column temperature, highly selective elution can be obtained. The thermodynamics of this temperature effect are different for the binding of specific and nonspecific DNA sequences, making chromatography at different temperatures a potentially important way of purifying transcription factors.
Jarrett
H W
HW
Department of Biochemistry, University of Tennessee, 858 Madison Avenue, Memphis, Tennessee, 38163, USA. hjarrett@utmem1.utmem.edu
D001426:Bacterial Proteins / Q000302:isolation & purification; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D005069:Evaluation Studies as Topic; D005656:Fungal Proteins / Q000302:isolation & purification; D049452:Green Fluorescent Proteins; D056968:Lac Repressors; D008164:Luminescent Proteins / Q000302:isolation & purification; D009687:Nuclear Proteins / Q000302:isolation & purification; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D012097:Repressor Proteins / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D014157:Transcription Factors / Q000302:isolation & purification*
D001426:Bacterial Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D056968:Lac Repressors; D008164:Luminescent Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D004247:DNA