Isolation of DNA-protein complexes based on streptavidin and biotin interaction.
We describe a method for the purification of proteins binding to specific DNA sites based on the strong interaction between streptavidin and biotin. We tested the efficiency of this method using the Escherichia coli lactose operon operator-repressor system. dUTP coupled to biotin is incorporated into a DNA fragment containing the lactose operator. A crude E. coli extract is first incubated with the biotinylated fragment and the reaction mixture is filtered on a streptavidin-agarose column. Proteins retained on the column are either eluted alone by high salt or isopropyl beta-D-thiogalactoside, or as a complex with the DNA site by enzymatic digestion of the DNA. We thus obtained a 3400-fold enrichment of the repressor complexed to the operator in one step. The method is simple and makes use of commercially available reagents. The large concentration of biotin-binding sites of the streptavidin-agarose matrix (0.1 mumol/ml packed gel) provides a very high capacity for the concentration and purification of large amounts of proteins. The advantage of this method for the detection and purification of other DNA-binding proteins is discussed.
Leblond-Francillard
M
M
Dreyfus
M
M
Rougeon
F
F
D001426:Bacterial Proteins / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004269:DNA, Bacterial / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D007763:Lac Operon; D008970:Molecular Weight; D012097:Repressor Proteins / Q000302:isolation & purification* / Q000378:metabolism; D019809:Streptavidin; D014157:Transcription Factors / Q000302:isolation & purification*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; D014157:Transcription Factors; D001710:Biotin; D019809:Streptavidin