Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems

This unit presents purification protocols that exploit the tight and essentially irreversible complex that biotin forms with streptavidin. A DNA fragment containing a high‐affinity binding site for the protein of interest is prepared and a molecule of biotinylated nucleotide is incorporated into one of the ends of the DNA fragment. The protein of interest is allowed to bind to the high‐affinity recognition site present in the biotinylated fragment. The tetrameric protein streptavidin is then bound to the biotinylated end of the DNA fragment. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently removed by adsorption onto a biotin‐containing resin. Since streptavidin is multivalent, it is able to serve as a bridge between the biotinylated DNA fragment and the biotin‐containing resin. Proteins remaining in the supernatant are washed away under conditions that maximize the stability of the DNA‐protein complex. Finally, the protein of interest is eluted from the resin with a high‐salt buffer. Both batch and column formats are presented, as is a protocol for the use of streptavidin‐agarose. A support protocol describes a mobility shift assay for detecting sequence‐specific DNA‐binding proteins.