Identification of amino acids essential for antibody binding by mRNA-display using a random peptide library: an anti-human tumor protein p53 antibody as a model.
Using a synthetic DNA library coding for random 10-amino acid peptides (R10aPL), mRNA-display was applied to the isolation of interactive peptides using a monoclonal antibody against human TP53 (hTP53) as a model. Display molecules consisting of peptides and the nucleotide sequences encoding them were synthesized in vitro and subjected to four to five cycles of affinity selection. Thirty-four clones each isolated in the 4th or 5th round were sequenced. A core sequence, (X)-S-D-L-(Z)-K-L essential for binding was found, in which (X) and (Z), though undefined, were mostly F or Y and W, respectively. Although no peptides that fully matched with hTP53 were found in the clones isolated, the core sequence was found in hTP53. A 10-amino acid peptide containing the core sequence was chemically synthesized to verify its binding with SPR. Its Kd value for the antibody was 6 nM. The amino acids in epitopes essential for binding could be identified by mRNA-display with R10aPL.
ZOEGENE Corporation Realigned with MOLECUENCE Corporation, Mitsubishi Chemical Group Yokohama Research Center, 1000 Kamoshida-cho, Aoba-ku, Yokohama, Kanagawa, 227-8502, Japan.
Miwa
M
Shiratori
Teruaki
T
Kobayashi
Tatsuro
T
Shibui
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology*; D000915:Antibody Affinity / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D020869:Gene Expression Profiling; D006801:Humans; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000235:genetics / Q000276:immunology*; D020539:Sequence Analysis, Protein; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D019151:Peptide Library; D010455:Peptides; D016159:Tumor Suppressor Protein p53