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Improved Protein–Protein Interaction Assay FlimPIA by the Entrapment of Luciferase Conformation
journal contribution
posted on 2014-02-18, 00:00 authored by Yuki Ohmuro-Matsuyama, Yuko Hara, Hiroshi UedaRecently we reported a novel protein–protein
interaction
assay FlimPIA (firefly luminescent intermediate-based protein–protein
interaction assay) based on the functional complementation of two
mutant firefly luciferases (Fluc), each defective in its one of two
half reactions. The assay detects approximation of two mutant Flucs,
namely, a “Donor” that catalyzes ATP-driven luciferin
adenylation to produce a luciferyl-adenylate intermediate, and an
“Acceptor” that mainly catalyzes subsequent oxidative
luminescent reaction. However, there was a problem in FlimPIA that
the remaining adenylation activity of the Acceptor constituted its
background signal and hampered its wider use. In this study, we aimed
at reducing the background signal by trapping the Acceptor to the
“oxidation” conformation, either chemically or by disulfide
bonding. The results showed higher sensitivity and detection over
the longer distance of the developed assay compared to conventional
FlimPIA, Fluc-based protein-fragment complementation assay, and fluorescent
protein-based FRET assay.