Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides.
We describe a rapid and sensitive method to isolate sets of high-affinity binding sites for sequence-specific DNA binding proteins. The DNA binding domain of the protein is expressed in Escherichia coli as a fusion with glutathione S-transferase (GST). Binding reactions are set up with total soluble extract from induced bacteria and a double-stranded oligonucleotide for which the central 32 bp have been randomized. To ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C). The GST fusion protein is recovered by the addition of glutathione-Sepharose. Following extensive washing of the Sepharose beads, the bound oligonucleotides are rescued by polymerase chain reaction amplification. The amplified material is used in the next cycle of selection and amplification. Approximately five cycles are needed to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form.
Department of Molecular Biology, Göteborg University, Sweden.
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Pierrou
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Enerbäck
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Carlsson
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D005982:Glutathione Transferase