title: Ultra sensitive firefly luciferase-based protein-protein interaction assay (FlimPIA) attained by hinge region engineering and optimized reaction conditions.
abstract: 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 Km 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.
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Kurihara
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.|Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan.|The Japan Society for the Promotion of Science, Tokyo, Japan.|Department of Applied Chemistry, School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Ohmuro-Matsuyama
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Keiichi
authors.initials: K
authors.lastname: Ayabe
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamashita
authors.affiliation: Department of Applied Chemistry, School of Engineering, Kobe University, Kobe, Japan.
authors.forename: Hideki
authors.initials: H
authors.lastname: Yamaji
authors.affiliation: Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Tokyo, Japan. ueda@res.titech.ac.jp.|Chemical Resources Laboratory, Tokyo Institute of Technology, Yokohama, Japan. ueda@res.titech.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Ueda
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D020134:Catalytic Domain; D003065:Coenzyme A / Q000378:metabolism; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D015202:Protein Engineering; D060066:Protein Interaction Maps
chemical_list: D000255:Adenosine Triphosphate; D049409:Luciferases, Firefly; D003065:Coenzyme A
keywords: ANL enzyme superfamily; Enzyme engineering; Firefly luciferase; Protein-protein interaction