Heparin elution of transcription factors from DNA-Sepharose columns.
A novel method using heparin for eluting transcription factors from DNA-Sepharose columns was characterized. CAAT enhancer binding protein (C/EBP) or lac repressor fusion proteins were both eluted with heparin from columns containing specific DNA sequences coupled to cyanogen bromide activated Sepharose. The amount of the lac repressor chimera which eluted from the column was shown to increase with increases in the mobile phase heparin concentration. The elution of the protein was also shown to be dependent on the amount of DNA coupled to the column and more protein eluted from columns containing lesser amounts of DNA. These data suggest that heparin and DNA compete for binding to the protein; this competition causes elution. Comparison of heparin- and salt-eluted protein demonstrated the heparin-eluted fraction was significantly purer and comparable to that obtained by elution with isopropyl beta-D-thiogalactopyranoside, a lactose analog. Heparin elution represents an important new tool in the purification of transcription factors and other DNA-binding proteins by DNA affinity chromatography.
Gadgil
H
H
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Jarrett
H W
HW
D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D004247:DNA; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins; D006493:Heparin / Q000737:chemistry*; D056968:Lac Repressors; D009687:Nuclear Proteins / Q000302:isolation & purification*; D012097:Repressor Proteins / Q000302:isolation & purification*; D012685:Sepharose; D014157:Transcription Factors / Q000302:isolation & purification*
D001426:Bacterial Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D006493:Heparin; D004247:DNA; D012685:Sepharose