IDENTIFYING PEPTIDE LIGANDS FOR BARLEY ALPHA-AMYLASE-1 USING COMBINATORIAL PHAGE DISPLAY LIBRARIES

A synthetic combinatorial Library of 15-mer peptides expressed as N-te rminal fusion to pill on the surface of filamentous bacteriophage was screened to identify specific ligands for barley alpha-amylase 1. Affi nity selection of phages that display tight-binding peptides was accom plished by four cycles of panning, using purified alpha-amylase enzyme as the immobilized binding target. The amino acid sequences of the ti ght-binding peptides were determined by sequencing the corresponding c oding region in the viral DNA genome. A total of 13 clones, randomly s elected from the final enriched Library, were found to have the follow ing sequences: TRWLVYFSRPYLVAT (8 clones), PRHVFYRWFLSNPRI (4 clones), and IVRHLFLHVYPRVLM (1 clone). Binding activity of affinity-purified phage clones was confirmed by ELISA and quantitated by PFU assay. All three peptides share a common feature of an Arg residue flanked by hig h numbers of Tyr and Trp/Phe residues. The phage display peptide TRWLV YFSRPYLVAT showed an activity 5-fold greater than those of the other t wo peptides and a dissociation constant of 4.4 x 10(-9) M.