AFFINITY SELECTIVE ISOLATION OF LIGANDS FROM PEPTIDE LIBRARIES THROUGH DISPLAY ON A LAC REPRESSOR HEADPIECE DIMER

DNA binding by the Escherichia coli lac repressor is mediated by the s imilar to 60 amino acid residue 'headpiece' domain. The dimer of headp iece domains that binds to the lac operator is normally formed by asso ciation of the much larger similar to 300 amino acid residue C-termina l domain. We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short pept ide linker. These proteins bind plasmid molecules with sufficient stab ility to allow association of a peptide epitope displayed at the C ter minus of the headpiece dimer with the plasmid encoding that peptide. L ibraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment o f peptide-headpiece dimer-plasmid complexes using an immobilized recep tor. After each round of enrichment, transformation of E. coli with re covered plasmids permits amplification of the selected population. Aft er several rounds of enrichment, sequencing of individual clones revea ls the structure of the selected peptides. Headpiece dimer libraries a llow selection of peptide ligands of higher average affinity than simi lar libraries based on the intact lac repressor. Interestingly, the pr esence of the lac operator is not required for plasmid binding by the headpiece dimer protein. (C) 1996 Academic Press Limited