Yuki Ohmuro-Matsuyama

By Yuki Ohmuro-Matsuyama and Hiroshi Ueda

Protein-fragment complementation assays (PCAs) are commonly used to assay protein–protein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we developed a novel protein–protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more stable probes, and higher signal readout in a shorter time period, and it also worked in cellulo.

Part of the book: Protein-Protein Interaction Assays

By Yuki Ohmuro-Matsuyama and Hiroshi Ueda

Protein-protein interaction assays are fundamental to basic biology, drug discovery, diagnostics, screening, and immunoassays. Protein-fragment complementation (PCA) is one of such useful protein-protein interaction assays. PCA when performed using luciferase is a reversible approach, whereas when performed using green fluorescent protein analogs is an irreversible approach. The NanoLuc technology developed in 2012 utilizes a small and structurally robust luciferase that is capable of producing very bright luminescence. NanoLuc PCA has been used to detect many protein-protein interactions and for screening purposes. Methods developed from NanoLuc PCA include the HiBiT technology and NanoLuc ternary technology. These novel technologies are promising in various fields and further developments are anticipated.

Part of the book: Bioluminescence