Preparation and use of a universal primed Sepharose for the purification of DNA-binding proteins

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作者：

B Arcangioli，S Pochet，R Sousa，T Huynh-Dinh

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摘要：

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 Xho I 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 Xho I 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.

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关键词：

binding sites, protein biochemistry, gels, DNA, purification, nucleotide sequences, binding proteins, electrophoresis, polyacrylamide, Saccharomyces cerevisiae

DOI：

10.1111/j.1432-1033.1989.tb14561.x

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年份：

2010