**1. Introduction**

When the human genome project was completed in 2003, most researchers expected dramatic developments in various fields such as biology, etiology, and drug discovery. However, progression did not remarkably accelerate. One of the causes is that protein-protein interactions (PPIs) are still not well understood. In the cell, many proteins interact with each other and cooperate to fulfill their roles in biological phenomena. It is reported that there are 150,000– 300,000 PPIs in the human interactome [1, 2]. Therefore, PPI assays are very important for biology, diagnosis, and drug discovery.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The conventional PPI assays, which are available both in vitro and in cellulo, are Förster/fluorescence resonance energy transfer (FRET)-based assays, bioluminescent resonance energy transfer (BRET) assays, and protein-fragment complementation assays (PCAs).

For FRET-based assays, two fluorescent proteins or two fluorescent dyes are fused to proteins that interact with each other. When the interaction occurs, the two fluorescent proteins (dyes) are in close proximity, and then the energy transfer is induced, resulting in changes of the fluorescent intensities. In BRET assays, a bioluminescent enzyme and fluorescent protein (dye) are fused to proteins that interact with each other, and the energy is transferred from the bioluminescent enzyme to the fluorescent protein (dye). FRET- and BRET-based assays are the most common and sophisticated methods.

For PCA, the enzyme or fluorescence is divided into two fragments. The split fragments are fused to interacting proteins. Upon interaction, the split fragments come close, and then the full length of the structure is reconstituted, resulting in the recovery of the enzyme activity or fluorescence. PCA in cells and lysates is a user-friendly method that gives a high signal/ background (S/B) ratio [3]. Moreover, we reported in vitro PCA using purified firefly luciferase (Fluc) fragments for the first time [4, 5]. The development of PCA is described in Section 2.

Recently, we developed a novel PPI assay, named firefly luminescent intermediate-based proteinprotein interaction assay, FlimPIA [6–10]. FlimPIA utilizes the unique reaction of Fluc, which is divided into two half steps. We describe the principle of FlimPIA in Section 3.1 and the several improvements of FlimPIA in Sections 3.2–3.6. Then, the advantages and disadvantages of FlimPIA compared to another PPI assays such as the in vitro PCA are described in the final section.
