title: Two-stage selection of sequences from a random phage display library delineates both core residues and permitted structural range within an epitope.
abstract: Libraries of random peptides can be screened to identify species which interact with antibodies or receptors. Similarly, maps of native molecular interactions can frequently be deduced by screening a limited set of peptide fragments derived from sequences within a native antigen or ligand. However, the existence of cross-reactive sequences that mimic original epitopes and the limited replaceability of amino acid residues suggest that the sequence space accessible by a receptor can be much broader. Definition of this space is of particular importance where structural information is required for peptidomimetic or drug design. We have used a two-stage selection scheme to expand the sequence space accessible by a phage display library and to define peptide epitopes of the anti-FLAG octapeptide monoclonal M2 antibody. Affinity selection of a primary library of 2 x 10(6) random decapeptides identified a non-contiguous core of three residues in the binding motif Tyr-Lys-Xaa-Xaa-Asp. A second stage library with 2 x 10(7) individual clones bearing the core motif but with the remaining flanking and internal residues re-randomized permitted access to a broader sequence space represented in a library equivalent to several orders of magnitude larger. Data here demonstrate that extended access to binding sequence space permitted by multi-stage screening of phage display libraries can reveal not only essential residues required for ligand binding, but also the ligand structural range permitted within the receptor binding pocket.
authors.affiliation: Upjohn Company, Kalamazoo, MI 49007.
authors.forename: R M
authors.initials: RM
authors.lastname: Miceli
authors.forename: M E
authors.initials: ME
authors.lastname: DeGraaf
authors.forename: H D
authors.initials: HD
authors.lastname: Fischer
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D017911:Inoviridae / Q000235:genetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000276:immunology*; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012641:Selection, Genetic
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide