ファージ・ディスプレイ法による抗体の分子進化とその応用

抄録

  Advances in methods for conformational prediction, structural analysis and site-directed mutagenesis of proteins and peptides have contributed to the understanding of their structure and function. However, with the exception of a few successes, the generation of practical functional molecules solely by rational design remains a difficult challenge. The aim of our study is to investigate molecular design relying on evolutionary processes, called as “directed evolution”, to generate a novel class of biofunctional molecules. This evolutionary approach consists of three steps; 1) constructions of protein/peptide libraries based on structural information, 2) expressions of the libraries on phage particles, and 3) selections with investigator-imposed selective pressures. In this work, we study on directed evolution with antibody libraries. We have succeeded in generating highly active catalytic antibodies in phage-displayed antibody (Fab) libraries. To evolve catalytic antibodies toward higher catalytic activity, we have mimicked an enzyme-evolutional process, in which an enzyme has evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. Thus, phage-displayed libraries derived from an original catalytic antiboby were selected against a newly-devised TSA, which was programmed to optimize the differential affinity for the transition state relative to the ground state, to provide variants with improved reaction rates (k_cat). The in vitro evolution has great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes.