[Directed evolution of antibody molecules in phage-displayed combinatorial libraries].
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 antibody 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.
Fujii
Ikuo
I
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Gakuen-cho, Sakai City, Osaka, Japan. fujii@b.s.osakafu-u.ac.jp
D000906:Antibodies / Q000235:genetics*; D017151:Antibodies, Catalytic; D019020:Directed Molecular Evolution / Q000379:methods*; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*
D000906:Antibodies; D017151:Antibodies, Catalytic; D019151:Peptide Library