Selection of a high affinity angiogenin-binding peptide from a peptide library displayed on phage coat protein

Although the phage-displayed peptide libraries have been widely used for the biopanning of peptide specific for various types of target molecules, the selected peptides often have affinities too low for practical purposes. In this report, selection of a high affinity peptide ligand specific for human angiogenin is described. We constructed a random dodecapeptide library displayed on the gene III protein of filamentous bacteriophage by use of a phagemid. The peptide insert was flanked by two cysteines to constrain the peptide structure by a disulfide bond. Phages were collected from 1.5 x 10(3) independent transformants. After the library phages were allowed to bind to the human angiogenin coated on a plate, the phages bound to the actin-binding site of angiogenin were selectively eluted with actin. The activity of each phage clone was estimated and three high affinity phage clones were identified. The peptides displayed by the three phage clones were synthesized as fusion proteins with Escherichia coli maltose-binding protein, and used for the quantitative estimation of their affinities. By this procedure, we were able to select a peptide having a dissociation constant of about 60 nM.