進化分子工学による人工酵素の設計と創出:Generation of Artifi cial Enzymes in Directed Evolution.
Ikuo Fujii, DOJIN News, 134 (2010)@font-face { font-family: “Times”; }@font-face { font-family: “ＭＳ 明朝”; }@font-face { font-family: “@ＭＳ 明朝”; }p.MsoNormal, li.MsoNormal, div.MsoNormal { margin: 0mm 0mm 0.0001pt; text-align: justify; font-size: 12pt; font-family: “Times New Roman”; }div.Section1 { page: Section1; }
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 generation of artificial biocatalysts by immune system that uses the evolutionary processes to give receptor-like molecules. As a natural enzyme binds the transition state of the chemical reaction to lower the activation energy, immunization with a putative transition-state analog (TSA), with the expectation that the induced antigen-combining site could be both geometrically and electronically complementary to the transition-state, provides catalytic antibodies. We have succeeded to generate antibodies catalyzing highly stereo and regio-specific hydrolyses with immunization of phosphonate transition-state analogs. Furthermore, to evolve catalytic antibodies toward higher catalytic activity, phage-displayed antibody (Fab) libraries were constructed to screen antibodies with optimized differential affinity for the transition state relative to the ground state. Recently, we have demonstrated a new strategy for generating catalytic antibodies, namely, by the generation of antigen-combining site that function as an apoprotein for binding functionalized components, called “holoabzyme”. Replacement of the functionalized components enables a single antibody to catalyze multiple reactions, β-elimination, decarboxylation and aldol reactions. Catalytic antibodies have great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes.