A Protein–Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases
There is a significant focus on detecting and assaying protein–protein interactions (PPIs) in biology and biotechnology. Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI by splitting the enzyme-coding or fluorescent protein-coding polypeptide, as well as Förster resonance energy transfer (FRET). Here, we describe a novel PPI assay FlimPIA (firefly luminescent intermediate-based protein–protein interaction assay) by a unique approach of splitting the two major catalytic steps (half reactions) of firefly luciferase (FLuc).
Key wordsProtein–protein interaction Firefly luciferase Substrate channeling Reaction intermediate Luciferyl adenylate
We thank B. Branchini for providing inspiration for the project. This project was supported by SENTAN, JST, Japan, and partly by the “Leave a Nest” Microtech Nichion award.
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