Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVRrecognizing the Puumala virus (PUUV) G2-glycoprotein-specificneutralizing monoclonal antibody (MAb) 1C9 with K_d of 2.85x10^–8from a random peptide library X₂CX₁₄CX₂ expressed on the pIIIprotein of the filamentous phage fd-tet. We have now createda second-generation phage-displayed peptide library in whicheach amino acid of the peptide was mutated randomly to anotherwith a certain probability. Peptides were selected for higheraffinity for MAb 1C9 and for a common binding motif for MAb4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein.The resulting peptides were synthesized as spots on cellulosemembrane. Amino acid changes which improved the reactivity ofthe peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCALwith K_d of 1.49x10^–9 in biosensor measurements. Our resultsshow that the binding properties of peptides, the affinity andthe specificity can be improved and the binding specificitydetermining amino acids and structural factors can be analyzedby combining binding assays with synthetic peptides on membranewith the use of second-generation phage display libraries. Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.