Purification of the c-fos enhancer-binding protein.
We have purified the c-fos enhancer-binding protein from HeLa cell nuclear extracts. The key purification steps involved chromatography on a nonspecific DNA affinity column, from which binding activity and other protein were eluted at low salt concentrations, followed by chromatography on a specific oligonucleotide affinity column, from which the enhancer binding activity was specifically eluted at high salt concentrations. The purified protein had a strong affinity for the c-fos enhancer dyad symmetry sequence, with an equilibrium dissociation constant of 3.3 x 10(-11) M. This affinity was at least 50,000-fold stronger than that found for nonspecific DNA sequences.
Prywes
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Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
Roeder
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RG
D002845:Chromatography; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000302:isolation & purification*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D014157:Transcription Factors / Q000302:isolation & purification*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D003850:Deoxyribonuclease I