title: Affinity purification of sequence-specific DNA binding proteins.
abstract: We describe a method for affinity purification of sequence-specific DNA binding proteins that is fast and effective. Complementary chemically synthesized oligodeoxynucleotides that contain a recognition site for a sequence-specific DNA binding protein are annealed and ligated to give oligomers. This DNA is then covalently coupled to Sepharose CL-2B with cyanogen bromide to yield the affinity resin. A partially purified protein fraction is combined with competitor DNA and subsequently passed through the DNA-Sepharose resin. The desired sequence-specific DNA binding protein is purified because it preferentially binds to the recognition sites in the affinity resin rather than to the nonspecific competitor DNA in solution. For example, a protein fraction that is enriched for transcription factor Sp1 can be further purified 500- to 1000-fold by two sequential affinity chromatography steps to give Sp1 of an estimated 90% homogeneity with 30% yield. In addition, the use of tandem affinity columns containing different protein binding sites allows the simultaneous purification of multiple DNA binding proteins from the same extract. This method provides a means for the purification of rare sequence-specific DNA binding proteins, such as Sp1 and CAAT-binding transcription factor.
authors.forename: J T
authors.initials: JT
authors.lastname: Kadonaga
authors.forename: R
authors.initials: R
authors.lastname: Tjian
mesh_terms: D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D012685:Sepharose
chemical_list: D004268:DNA-Binding Proteins; D007202:Indicators and Reagents; D004247:DNA; D012685:Sepharose