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.