Single-chain 434 repressors with altered DNA-binding specificities - Isolation of mutant single-chain repressors by phenotypic screening of combinatorial mutant libraries

Combinatorial mutant libraries of the single-chain 434 repressor were used to discover novel DNA-binding specificities. Members of the library contain one wild type domain and one mutant domain which are connected by a recomb inant peptide linker. The mutant domain contains randomized amino acids in place of the DNA-contacting residues. The single-chain derivatives are expe cted to recognize artificial operators containing the DNA sequence of ACAA - 6 base-pairs - NNNN, where ACAA is bound by the wild-type and NNNN by the mutant domain. An invivo library screening method was used to isolate muta nt DNA-binding domains which recognize the TTAA site of an asymmetric opera tor. Several mutants showed high affinity binding to the selection target a nd also strong (up to 80 fold) preference for TTAA over the wild type TTGT sequence. Some of the isolated mutants bound with very high affinities (10- 50 pM) to operators containing the TTAC sequence, a close homologue of the TTAA selection target.