Probing protein-protein interactions in living cells is crucial for understanding the protein functions and drug development. Small-sized protein binders are considered effective and useful for such analysis. Here we describe the development and use of a repebody, which is a protein binder composed of LRR (Leucine-rich repeat) modules, for tracking protein-protein interaction and localization in real-time. We developed an anti-mRFP repebody through a phage display method. Moreover, we designed an efficient and an EGFR-specific delivery platform using repebody to transport protein into the cells.
In Chapter 1, a repebody with high affinity for a red fluorescent protein was selected through a phage display, fused with a green fluorescent protein, and applied for tracing a red fluorescent protein-fused target protein in mammalian cells. We have also demonstrated that a repebody can be effectively used for the analysis of protein-protein interaction in living cells by live-cell imaging.
Chapter 2 describes the development of EGFR-specific repebody based delivery platform for receptor-specific intracellular delivery of cargos. We have shown that intracellular delivery of green ？uorescent protein (GFP) and gelonin toxin into the cytoplasm of mammalian cells. In consistent with an expression level of EGFR, repebody delivery system showed green fluorescence and cytotoxicity effects in mammalian cells in a dose dependent manner.
In this study, we successfully developed a repebody for targeting intracellular protein and repebody based delivery platform in living cells. Our results suggest that the developed repebodies can be widely used for the analysis of protein-protein interaction and an understanding of complex biological processes in living cells. Furthermore, the present approach of repebody immunotoxin can be a new therapeutic agent for the treatment of cancer.