Development of a repebody for analyzing and tracking protein-protein interactions

Abstract

Protein-protein interactions (PPIs) are biological processes for cell metabolism, membrane transport, and signal transduction. Based on the fundamental roles in the cell, the importance of protein interactions has been increasing in studies of both basic researches and human diseases. To date, many methods to analyze specific protein interactions have been used through conventional antibodies and natural protein binders. In this regard, we developed new protein binders for analyzing and tracking protein-protein interactions. In chapter 1, we used a non-antibody scaffold to detect and quantify human tumor necrosis factor-alpha (hTNF- $alpha$) for immunoassays. We selected specific binder through phage display and modular engineering. Selected binder was genetically fused to monomeric alkaline phosphatase (mAP), and the resulting mAP fusion protein was used for direct and sandwich immunoassays of hTNF- $alpha$. mAP fusion protein exhibited high sensitivity and accuracy in a direct and sandwich immunoassays. Furthermore, a monomeric AP was more suitable as a signal generator than a dimeric AP in an immunoassay. Chapter 2 describes development of a novel protein binder to detect and analyze human Rac1 (Ras-related small GTPase). We selected a novel Rac1 binder with a high binding affinity and specificity, and selected binders exhibited a higher selectivity to active Rac1 than inactive Rac1. A selected binder completely inhibited effector protein binding to active Rac1, which indicates potential utility in cancer therapy. Through cell-based assay, we confirmed that selected binder can effectively detect endogenous Rac1 using protein delivery system and analyze cell movement using expression of Rac1 binder in mammalian cells. In this study, we demonstrated the utility and potential of a non-antibody scaffold by showing development of hTNF-$alpha$ for immunoassays and analysis of human Rac1. These results indicate that target specific binders can be widely used in methods for detection and analysis of protein-protein interactions while alternating conventional antibodies and natural protein binders.