Identification of a structural epitope by using a peptide library displayed on filamentous bacteriophage.
The screening of phage-displayed random peptide libraries has recently emerged as a powerful technique for probing Ab-Ag interactions. We have used this method to identify the epitope recognized by a mAb, CB5B10, raised against plasminogen activator inhibitor type-1 (PAI-1). Two phage libraries, displaying random hexapeptides with or without flanking cysteine residues, were screened for binding to mAb CB5B10. The selected phages were shown to contain similar peptide sequences, all of which were flanked by cysteines. When compared with the crystal structure of PAI-1, the selected peptides closely resemble the sequence of a solvent-exposed loop connecting the COOH-terminal of an alpha-helix at Phe114 to a beta-sheet at Ser119. Because of the constraints imposed by the flanking cysteine residues, the selected peptides appear to mimic the structure and the sequence of the PAI-1 epitope. Specific contacts between the amino acids displayed by the phage and the mAb were explored using site-directed mutants of the phage peptide. The effects of these substitutions on binding to the mAb correlated well with the accessibility of the corresponding residues in the PAI-1 epitope. This is the first example of the use of phage-displayed peptide libraries to identify a structural epitope.
Hoess
R H
RH
DuPont Merck Pharmaceutical Company, Wilmington, DE 19880.
Mack
A J
AJ
Walton
H
H
Reilly
T M
TM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001435:Bacteriophages / Q000235:genetics*; D001483:Base Sequence; D000939:Epitopes / Q000032:analysis*; D005260:Female; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D017395:Plasminogen Activator Inhibitor 1