49 - Magnetic DNA Affinity Purification of Yeast Transcription Factor

Abstract

This chapter describes the preparation of magnetic DNA affinity beads and their use in the purification of sequence-specific DNA-binding proteins. The protein chosen for illustration of the method is yeast transcription factor τ, one of the most complex transcription factors. For purification, a protein fraction containing the sequence-specific DNA-binding proteins is incubated with DNA affinity beads using a ratio of specific protein to immobilized DNA that nearly saturates the affinity beads. During competition between specific and nonspecific DNA-binding proteins for limited amounts of sites, the specific protein is favored if it has the strongest affinity for the recognition sequence immobilized on the beads. DNA harboring the specific recognition sequence can be either in the form of a DNA fragment prepared by restriction enzyme digestion of a plasmid, or in the form of a duplex oligonucleotide, preferentially polymerized, or the product of the scaled-up polymerase chain reaction using biotinylated oligonucleotide primers. The protein, yeast transcription factor τ interacts with a split intragenic promoter and protects more than eight helical turns of DNA. Because the preparation of DNA affinity beads for protein purification requires the use of milligram amounts of plasmid, a large-scale plasmid preparation is needed.