Selection of sugar-binding peptides from a scaffold-containing library

Molecular evolution of binding affinity of artificial peptides by random mutation and affinity selection was performed. DNA fragments encoding ganglioside Galβ1-3GalNAcβ1-4(Neu5Acα2-3)Galβl-4Glcβ1- 1′Cer (GM1)-binding peptides were amplified by error-prone polymerase chain reaction with single strand DNA of phages. This peptide contains artificial zinc-binding sequence to restrict peptide conformation. The amplified DNA fragments were digested with Acc65 I and Eag I, and the fragment was ligated into the M13KE phagemid vector. The vector was used to transform host cells (ER2738) by electroporation and the corresponding phage particles were generated. After two cycles of an affinity selection against GM1 monolayer, three positive phage clones were isolated. Phage ELSIA indicated that these clones had affinity for GM1 at 0.01 nM. The binding affinity of an ee2-1 mutant, ee2-15, altered at His-3 to Leu was found to be enhanced. We showed the binding affinity of peptides for target sugar chain was improved by molecular evolution process.