Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library.
We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVR recognizing the Puumala virus (PUUV) G2-glycoprotein-specific neutralizing monoclonal antibody (MAb) 1C9 with Kd of 2.85 x 10(-8) from a random peptide library X2CX14CX2 expressed on the pIII protein of the filamentous phage fd-tet. We have now created a second-generation phage-displayed peptide library in which each amino acid of the peptide was mutated randomly to another with a certain probability. Peptides were selected for higher affinity for MAb 1C9 and for a common binding motif for MAb 4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein. The resulting peptides were synthesized as spots on cellulose membrane. Amino acid changes which improved the reactivity of the peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCAL with Kd of 1.49 x 10(-9) in biosensor measurements. Our results show that the binding properties of peptides, the affinity and the specificity can be improved and the binding specificity determining amino acids and structural factors can be analyzed by combining binding assays with synthetic peptides on membrane with the use of second-generation phage display libraries.
Heiskanen
Tuomas
T
Department of Virology, Haartman Institute, P.O. Box 21, FIN-00014 University of Helsinki, Finland. tuomas.heiskanen@helsinki.fi
Li
Xiao-Dong
XD
Hepojoki
Jussi
J
Gustafsson
Elisabeth
E
Lundkvist
Ake
A
Vaheri
Antti
A
Lankinen
Hilkka
H
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001665:Binding Sites; D003001:Cloning, Molecular; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006480:Hemorrhagic Fever with Renal Syndrome / Q000097:blood / Q000276:immunology / Q000821:virology; D006801:Humans; D018716:Molecular Mimicry; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology; D019151:Peptide Library; D011487:Protein Conformation; D029262:Puumala virus / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D000939:Epitopes; D010446:Peptide Fragments; D019151:Peptide Library; D014759:Viral Envelope Proteins