Improved sensitivity of firefly luminescent intermediate‐based protein interaction assay using Ser 440 mutant with lower adenylation activity.

Abstract: Protein–protein interaction assays are important in various fields including molecular biology, diagnostics, and drug screening. We recently designed a novel protein–protein interaction assay, the firefly luminescent intermediate‐based protein interaction assay (FlimPIA), that exploited the unique reaction mechanism of firefly luciferase (Fluc). Using two mutant Flucs, each impaired with one of the two half‐reactions, namely adenylation and subsequent oxidative luminescent steps, FlimPIA detects the proximity of the two proteins tethered to the mutant Flucs. Here, we found that introducing a mutation into a residue in the hinge region (S440) of the mutant with lowered adenylation activity (‘Acceptor’ Fluc) further improved the response of FlimPIA by lowering the residual adenylation activity. Mutants with bulkier residues showed greater inhibition, probably due to increased steric hindrance at the adenylation conformation. As a result, the FlimPIA with S440 L acceptor showed the best signal/background ratio for the detection of rapamycin‐induced FKBP12–FRB interactions. [ABSTRACT FROM AUTHOR]