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-affin ity binding sites for sequence-specific DNA binding proteins, The DNA binding domain of the protein is expressed in Escherichia coli as a fu sion with glutathione S-transferase (GST). Binding reactions are set u p with total soluble extract from induced bacteria and a double-strand ed oligonucleotide for which the central 32 bp have been randomized. T o ensure stringent conditions, binding is done in the presence of high levels of poly(dI:C), The GST fusion protein is recovered by the addi tion of glutathione-Sepharose, Following extensive washing of the Seph arose beads, the bound oligonucleotides are rescued by polymerase chai n reaction amplification. The amplified material is used in the next c ycle of selection and amplification, Approximately five cycles are nee ded to obtain a pure population of high-affinity sites, which are then cloned and sequenced. This procedure should be applicable to any sequ ence-specific DNA binding protein for which the cDNA is available and which can be expressed in bacteria in a functional form. (C) 1995 Acad emic Press, Inc.