Design of affinity-controllable protein binder based Leucin-rich-repeat (LRR) modules

Abstract

Many proteins exert their biological roles as components of complexes, and the functions of proteins are often determined by their specific interactions with other proteins. Therefore, the ability to control protein-protein interactions provides a powerful tools for molecular diagnostics, cell-based biosensors, therapeutics, and industrial biotechnology. In this study, we developed affinity-controllable protein binder based leucine-rich-repeat (LRR) module protein. Chapter 1 describes the development of pH-sensitive protein binder and its application for affinity purification of human immonuglobulin antibodies. The importance of a downstream process for the purification of antibodies is increasing with the growing application of monoclonalantibodies in many different areas. Althogh protein A is the most commonly used for the affinity purification of antibodies, certain properties could be further improved: milder elution condition and higher stability in alkaline solution. Herein, we present the development of Fc-specific repebody by modular engineering approach and its potential as an affinity ligand for purification of human IgG. The developed repebody was shown to be loss of binding affinity at mild pH (pH 4.0). Also, the repebody remained almost intact even after incubation with harsh pH conditions. In Chapter 2, we have first demonstrated photo-switchable scaffold protein using photo-isomerizable ligand. Photo-switchable proteins are powerful tools to control biological processes with the spatial and temporal precision of light. Unfortunately, such proteins exist only for specific cases of proteins and general ligand which can bind variable targets has not developed. Here, we describe the development of photo-switchable repebody by computational design and its potential as a light-controlled binder. We expect that developed photo-switchable repebody scaffold can be used to the robust and general application for light-controlled biological studies.