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
2009
Shiratori, Miwa | Kobayashi, Teruaki | Shibui, Tatsuro
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 K d value for the antibody was 6 nM. The amino acids in epitopes essential for binding could be identified by mRNA-display with R10aPL.
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