Cm. Gates et al., AFFINITY SELECTIVE ISOLATION OF LIGANDS FROM PEPTIDE LIBRARIES THROUGH DISPLAY ON A LAC REPRESSOR HEADPIECE DIMER, Journal of Molecular Biology, 255(3), 1996, pp. 373-386
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