Identifying Peptide Ligands for Barley α-Amylase 1 Using Combinatorial Phage Display Libraries

Abstract

A synthetic combinatorial library of 15-mer peptides expressed as N-terminal fusion to pIII on the surface of filamentous bacteriophage was screened to identify specific ligands for barley α-amylase 1. Affinity selection of phages that display tight-binding peptides was accomplished by four cycles of panning, using purified α-amylase enzyme as the immobilized binding target. The amino acid sequences of the tight-binding peptides were determined by sequencing the corresponding coding region in the viral DNA genome. A total of 13 clones, randomly selected from the final enriched library, were found to have the following 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 high numbers of Tyr and Trp/Phe residues. The phage display peptide TRWLVYFSRPYLVAT showed an activity 5-fold greater th...