A catalysis-based selection for peroxidase antibodies with increased activity.
A biotin-tyramine conjugate (1) was found to covalently cross-link with peroxidase antibody 7G12 upon the catalytic oxidation of the tyramine moiety in the presence of hydrogen peroxide (H2O2). On the basis of this observation, a novel strategy was developed to select mutants of 7G12 Fab with enhanced peroxidase activity from a library of phage displayed antibodies. In such a selection, tyramine is oxidized by hydrogen peroxide in a process catalyzed by peroxidase antibodies displayed on phage. Antibodies with higher peroxidase activity are preferentially labeled with biotin through irreversible adduct formation between oxidized biotin-linked tyramine molecules and phenolic side chains of the antibody. The corresponding phage particles can then be selected via biotin-streptavidin interactions. Using this strategy, phage displayed libraries of antibody 7G12 were selected for higher peroxidase activity. As a result, mutations of antibody 7G12 that led to 10 to 20-fold increases in the peroxidase activity (kcat/Km) were identified, suggesting the validity of this method for the evolution of peroxidase antibodies based directly on catalytic turnover.
Yin
Jun
J
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Mills
Jeremy H
JH
Schultz
Peter G
PG
D017151:Antibodies, Catalytic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017104:Bacteriophage M13 / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D002384:Catalysis; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008652:Mesoporphyrins / Q000737:chemistry* / Q000276:immunology; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D019151:Peptide Library; D010544:Peroxidases / Q000737:chemistry* / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry
D017151:Antibodies, Catalytic; D007128:Immunoglobulin Fragments; D008652:Mesoporphyrins; D019151:Peptide Library; C065420:N-methylmesoporphyrin IX; D006418:Heme; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D010544:Peroxidases