Open AccessPurification of the c-fos enhancer-binding protein.
Author(s) -
Ron Prywes,
Robert G. Roeder
Publication year - 1987
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.7.10.3482
Subject(s) - enhancer , biology , affinity chromatography , dissociation constant , dna , oligonucleotide , elution , microbiology and biotechnology , dna binding protein , biochemistry , chromatography , transcription factor , chemistry , gene , enzyme , receptor
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.