Purification of DNA-binding proteins using biotin/streptavidin affinity systems.
Short fragments of DNA-either natural or formed from oligonucleotides-containing a high-affinity site for a DNA-binding protein provide a powerful tool for purification. The biotin/streptavidin purification system is based on the tight and essentially irreversible complex that biotin forms with streptavidin. In this procedure, a DNA fragment is prepared that contains a high-affinity binding site for the protein of interest, and a molecule of biotinylated nucleotide is then incorporated into one of the ends of the DNA fragment. The protein of interest binds to the DNA, and then this complex binds (via the biotin moiety) to the tetrameric protein streptavidin. Next, the protein/biotinylated fragment/streptavidin ternary complex is efficiently isolated 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 removed by washing, and the resin-bound protein is then eluted with a high-salt buffer. An alternate protocol describes a microcolumn method that is useful for larger volumes of biotin-cellulose resin. This method is also used to elute the protein in as small a volume (i.e., as high a concentration) as possible. Another variation on the basic procedure is provided in which streptavidin-agarose is employed.
Chodosh
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Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
D000818:Animals; D001426:Bacterial Proteins; D001710:Biotin; D019921:Biotinylation; D002482:Cellulose / Q000031:analogs & derivatives; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D011485:Protein Binding; D012685:Sepharose / Q000031:analogs & derivatives; D019809:Streptavidin
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; C049538:streptavidin-agarose; D001710:Biotin; D002482:Cellulose; D004247:DNA; D012685:Sepharose; D019809:Streptavidin