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Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_8, © Springer Science+Business Media LLC 2017
Chapter 8
Ultrasensitive Firefly Luminescent Intermediate-Based
Protein-Protein Interaction Assay (FlimPIA) Based
on the Functional Complementation of Mutant Firefly
Luciferases
Yuki Ohmuro-Matsuyama and Hiroshi Ueda
Abstract
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 (LH 2 -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.
Key words Protein-protein interaction, Firefly luciferase, Substrate channeling, Reaction intermediate, Sensitivity, Detectable distance, Signal-background ratio
1 Introduction
Development of protein-protein interaction (PPI) assays is considered highly important in various fields, such as molecular and
cellular biology, diagnostics, and drug screening. Recently, we
developed a novel protein-protein interaction assay that we termed
FlimPIA (Firefly luminescent intermediate-based Protein-protein
Interaction Assay) based on the following unique detection
principle [1].
Viktor Stein (ed.), Synthetic Protein Switches: Methods and Protocols, Methods in Molecular Biology, vol. 1596,
DOI 10.1007/978-1-4939-6940-1_8, © Springer Science+Business Media LLC 2017
Chapter 8
Ultrasensitive Firefly Luminescent Intermediate-Based
Protein-Protein Interaction Assay (FlimPIA) Based
on the Functional Complementation of Mutant Firefly
Luciferases
Yuki Ohmuro-Matsuyama and Hiroshi Ueda
Abstract
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 (LH 2 -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.
Key words Protein-protein interaction, Firefly luciferase, Substrate channeling, Reaction intermediate, Sensitivity, Detectable distance, Signal-background ratio
1 Introduction
Development of protein-protein interaction (PPI) assays is considered highly important in various fields, such as molecular and
cellular biology, diagnostics, and drug screening. Recently, we
developed a novel protein-protein interaction assay that we termed
FlimPIA (Firefly luminescent intermediate-based Protein-protein
Interaction Assay) based on the following unique detection
principle [1].