Isolation of DNA-protein complexes based on streptavidin and biotin interaction

Abstract

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 p-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 pmol/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.Gene transcription is regulated by both cis and transacting factors. Regulatory sequences located either near the transcription start site, such as promoters, or variably located such as enhancers, have been identified. Cell-type-specific transcription is presumably conferred by the interaction of selective proteins with certain of these sequences. Genetic methods, such as cell fusion or transfection experiments, provide an indirect approach in the study of trans-acting factors, while in vitro transcription assays [l], DNA protection 121, gel retardation [3, 41 and Western blotting [5] methods have yielded direct information on the interaction between sitespecific binding proteins and DNA regulatory sequences. A complete characterisation of these factors, however, necessarily depends on their purification. Since they are generally present at very low cellular concentration, powerful methods such as affinity chromatography using the target DNA must be employed.Several authors have recently reported different methods to obtain a DNA site matrix. Rosenfeld and Kelly [6] coupled a plasmid bearing multiple recognition sites to cellulose. Alternatively, Kadonaga and Tjian [7] used a polymerised synthetic oligonucleotide. Levens and Howley [8] fixed a / Igalactosidase -lac-repressor fusion protein to anti-(pga1actosidase)-Sepharose. The DNA sequence of interest was ligated to the lactose operator sequence and thus retained on the column. Kasher et al.[9] used biotin-cellulose and a biotinylated DNA fragment to obtain a DNA matrix via streptavidin bridges.To various degrees all these methods suffer from difficulties due to competition between non-specific DNAbinding proteins and the protein of interest for binding to the target DNA, and to inactivation of the purified protein. We present here a novel method designed to minimize these problems, uniting individual advantages of the preceding met...

Preparation and Biological Activity of Molecular Probes to Identify and Analyze Jasmonic Acid-binding Proteins

“…4) A JA-biotin conjugate would be a useful tool for the purification of JA-binding proteins, because a convenient purification system can be constructed by utilizing the interaction between the biotin moiety of a JA-biotin conjugate and avidine. 5) A JA-FITC conjugate would also be useful to identify and analyze the function of JA-binding proteins. 6) This will make it possible to observe the binding between this type of compound and JA-binding proteins.…”

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR

“…Biotinylated M-CFt-21 target DNA was stably immobilized onto a SA5 sensor chip by taking advantage from the streptavidin-biotin interaction [Leblond-Francillard et al, 1987] as elsewhere described [Feriotto et al, 1999]. In different experiments, 700-800 RU were bound to the sensor chip after 25 µl injection of 80 pmoles of the M-CFt-21 mer in HBS-EP ( Fig.…”

“…Blank subtractions were performed in all of the experiments by subtracting from the experimental sensorgrams the sensorgrams obtained by injections of buffers. In order to obtain an efficient capture of MCFt-21 target DNA onto the sensor chip, the well documented streptavidin-biotin interaction was employed [Leblond-Francillard et al, 1987]. The sensor chip SA5 was used in order to capture 5′-biotinylated M-CFt-21 oligonucleotide, injected over the surface.…”

Real-time multiplex analysis of four beta-thalassemia mutations employing surface plasmon resonance and biosensor technology

“…In order to obtain an efficient capture of 5 0 -biotinylated PCR products onto the sensor chip, the welldocumented streptavidin-biotin interaction was employed. 24 To this end, the sensor chip SA, precoated with streptavidin, was used. After pretreatment with three 10 ml pulses with 40 mM NaOH-1 M NaCl, three injections of 40 ml of HBS-EP, 0.5 M NaCl, containing PCR products were administered in different flow cells.…”