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 approximately 60 amino acid residue 'headpiece' domain. The dimer of headpiece domains that binds to the lac operator is normally formed by association of the much larger approximately 300 amino acid residue C-terminal domain. We have used in vitro selection to isolate 'headpiece dimer' molecules containing two headpiece domains connected via a short peptide linker. These proteins bind plasmid molecules with sufficient stability to allow association of a peptide epitope displayed at the C terminus of the headpiece dimer with the plasmid encoding that peptide. Libraries of peptides displayed on the C terminus of a headpiece dimer can be screened for specific receptor ligands by affinity enrichment of peptide-headpiece dimer-plasmid complexes using an immobilized receptor. After each round of enrichment, transformation of E. coli with recovered plasmids permits amplification of the selected population. After several rounds of enrichment, sequencing of individual clones reveals the structure of the selected peptides. Headpiece dimer libraries allow selection of peptide ligands of higher average affinity than similar libraries based on the intact lac repressor. Interestingly, the presence of the lac operator is not required for plasmid binding by the headpiece dimer protein.
Gates
C M
CM
Affymax Research Institute, Palo Alto, CA 94304, USA.
Stemmer
W P
WP
Kaptein
R
R
Schatz
P J
PJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D000939:Epitopes / Q000032:analysis; D029968:Escherichia coli Proteins; D015723:Gene Library; D056968:Lac Repressors; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D018000:Receptors, Peptide / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D008024:Ligands; D009842:Oligopeptides; D010455:Peptides; D018000:Receptors, Peptide; D012097:Repressor Proteins