Preparation and use of a universal primed Sepharose for the purification of DNA-binding proteins.
We have devised a novel method for the construction of a DNA affinity matrix and tested its use in the purification of a sequence-specific DNA-binding protein from the yeast Saccharomyces cerevisiae. The matrix was prepared in two steps: first, a palindromic oligonucleotide containing an XhoI cohesive end was covalently linked via its loop to a Sepharose matrix; second, directly to this 'universal' primed Sepharose was ligated a 37-bp oligonucleotide, with XhoI cohesive ends, containing the sequence of the upstream activation sequence 1 (UAS1) site of the yeast iso-1-cytochrome c (CYC1) gene. After fractionating a yeast crude extract through DEAE-cellulose, heparin ultrogel and Mono Q columns, a single pass through the affinity matrix allowed the purification to apparent homogeneity of the 120-kDa protein factor P, which is responsible for the binding to the UAS1 site.
Arcangioli
B
B
Groupe de Biologie Moléculaire de la Levure, Institut Pasteur, Paris, France.
Pochet
S
S
Sousa
R
R
Huynh-Dinh
T
T
D001483:Base Sequence; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis; D012685:Sepharose / Q000138:chemical synthesis*
D004268:DNA-Binding Proteins; D012685:Sepharose