Ultrasensitive Firefly Luminescent Intermediate-Based Protein-Protein Interaction Assay (FlimPIA) Based on the...

We recently developed a protein-protein interaction assay, FlimPIA (Firefly luminescent intermediate-based Protein-protein Interaction Assay) based on the catalytic mechanism of firefly luciferase (Fluc) that can be divided into two half-reactions: the adenylation step and the oxidative luminescent steps. We engineered two mutant Fluc enzymes named "Donor" and "Acceptor" where the oxidative luminescent activity of the Donor is almost eliminated and the adenylation activity of the Acceptor is suppressed. When the Donor and the Acceptor are each fused to one of two interacting partners, and put together to interact, the Donor and the Acceptor come sufficiently close such that the Acceptor can react with the luciferyl-adenylate intermediate (LH₂-AMP) produced by the Donor, and thus emit luminescence.FlimPIA can be used in vitro and in cultured cells. Owing to recent improvements, it has several advantages in terms of signal/background ratio, detectable size of interacting partner, and sensitivity over conventional protein-protein interaction assays based on Förster/fluorescence resonance energy transfer and protein-fragment complementation performed in vitro. Here, we describe a protocol to make use of the latest version of FlimPIA which shows even lower background and higher signal than previously described ones.