Arcangioli B, Pochet S, Sousa R, Huynh-Dinh T
Groupe de Biologie Moléculaire de la Levure, Institut Pasteur, Paris, France.
Eur J Biochem. 1989 Feb 1;179(2):359-63. doi: 10.1111/j.1432-1033.1989.tb14561.x.
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.
