Tendamistat as a scaffold for conformationally constrained phage peptide libraries.
The alpha-amylase inhibitor Tendamistat (Hoe-467), a 74 amino acid beta-sheet protein from Streptomyces tendae has been expressed on the surface of the filamentous bacteriophage M13. Phage displaying Tendamistat inhibit the hydrolysis of starch by alpha-amylase, indicating that the displayed protein is functional. The displayed Tendamistat has been used as a molecular scaffold for the presentation of constrained random peptides. Two loops, comprising residues 38 to 40 and 60 to 65 of Tendamistat, were randomized using PCR mutagenesis. Libraries of approximately 10(8) different mutant Tendamistat molecules were tested for binding to monoclonal antibody A8, which recognizes endothelin. After three cycles of biopanning, phage were isolated that specifically bound the monoclonal antibody. Loop swapping and alanine replacement mutagenesis indicated that residues in the 60 to 65 loop are responsible for binding to the monoclonal antibody. This work demonstrates the use of relatively small non-antibody protein scaffolds for the presentation of constrained random peptide sequences to select for novel binding molecules.
McConnell
S J
SJ
Dupont-Merck Pharmaceutical Company, Experimental Station E328/B33, Wilmington, DE 19880-0328, USA.
Hoess
R H
RH
D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D017104:Bacteriophage M13 / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001710:Biotin / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D016232:Endothelins / Q000276:immunology; D000939:Epitopes / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D004220:Disulfides; D016232:Endothelins; D000939:Epitopes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D001710:Biotin; D000516:alpha-Amylases; D000409:Alanine; C030526:tendamistate