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Ultra sensitive firefly luciferase‐based protein‐protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions
Author(s) -
Kurihara Makoto,
OhmuroMatsuyama Yuki,
Ayabe Keiichi,
Yamashita Takahiro,
Yamaji Hideki,
Ueda Hiroshi
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201500189
Subject(s) - luciferase , hinge , protein engineering , firefly protocol , chemistry , biophysics , materials science , biochemistry , biology , enzyme , engineering , structural engineering , zoology , transfection , gene
Detecting and assaying protein‐protein interactions are significant research procedures in biology and biotechnology. We recently reported a novel assay to detect protein‐protein interaction, i.e. firefly luminescent intermediate‐based protein‐protein interaction assay (FlimPIA) using two mutant firefly luciferases (Flucs), which complement each other's deficient half reaction. This assay detects neighboring of two mutant Flucs, namely, a ”Donor“ that catalyzes the adenylation of firefly luciferin to produce a luciferyl‐adenylate intermediate, and an ”Acceptor“ that catalyzes the subsequent light emitting reaction. However, its rather high background signal, derived from the remaining adenylation activity of the Acceptor, has limited its usefulness. To reduce this background signal, we introduced a mutation (R437K) into the hinge region of the Acceptor, while maintaining the oxidative activity. Interestingly, the signal/background (S/B) ratio of the assay was markedly improved by the addition of coenzyme A and reduction of the ATP concentration, probably due to reduced inhibition by dehydroluciferyl‐adenylate formed during the catalysis and an increased ATP‐based K m value of the Acceptor, respectively. As a result, a significantly improved maximal S/B ratio from 2.5 to ∼40 was attained, which promises wider use of the assay in in vitro diagnostics, drug discovery, and expanding our knowledge of various biological phenomena.