Affinity purification of sequence-specific DNA binding proteins.

TLDR

The study presents a fast, effective method for affinity purification of sequence‑specific DNA binding proteins. The protocol synthesizes recognition‑site oligodeoxynucleotides, couples them to cyanogen bromide‑activated Sepharose to create an affinity resin, then incubates protein fractions with competitor DNA and passes them through the resin, with tandem columns enabling simultaneous purification of multiple DNA‑binding proteins. The affinity resin selectively captures sequence‑specific DNA binding proteins, enabling 500‑ to 1000‑fold enrichment of Sp1 to ~90% homogeneity at 30% yield, and allows purification of rare proteins such as Sp1 and CAAT‑binding transcription factor.

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.