A Protein–Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases

Ohmuro‐Matsuyama, Yuki; Ueda, Hiroshi

doi:10.1007/978-1-4939-3813-1_10

Cited by 3 publications

(3 citation statements)

References 13 publications

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“…Fire y luciferase, which generates bioluminescence during the oxidative decarboxylation of D-luciferin in the presence of ATP, has been used for numerous applications such as measuring biomass and cellular conditions (Lundin 2000) and protein-protein interaction (Christopoulos and Chiu 1995;Ohmuro-Matsuyama and Ueda 2016), assaying ATP-related enzymes (Clarke 2005;Lundin 2000), examining Dluciferin-generating enzymes using peptide-modi ed luciferin or luciferin derivatives (Cali et al 2006;Liu et al 2005;Noda et al 2010;O'Brien et al 2008), and performing real-time ATP imaging (Grygorczyk et al 2019). The re y luciferase gene, luc, has been used as a reporter gene (Wood and Gruber 1996) as well as for bioimaging (Kaskova et al 2016;Yan et al 2019).…”

Section: Introductionmentioning

confidence: 99%

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Yawata

Noda²,

Shimomura³

et al. 2021

Biotechnol Lett

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ObjectivesFire y luciferase, one of the most extensively studied enzymes, has numerous applications. However, luciferase activity is inhibited by sodium chloride. This study aims to expand the applications of re y luciferase in the presence of sodium chloride. ResultsWe rst obtained two mutant luciferase enzymes whose inhibition were alleviated and identi ed these mutations as Val288Ile and Glu488Val. Under dialysis condition (140 mM sodium chloride), the wild type was inhibited to 44% of its original activity level. In contrast, the single mutants, Val288Ile and Glu488Val, retained 67% and 79% of their original activity, respectively. Next, we introduced Val288Ile and Glu488Val mutations into the wild-type luciferase to create a double mutant using site-directed mutagenesis. Notably, the double mutant retained its activity more than 95% of that in the absence of sodium chloride. ConclusionsThe mutant luciferase, named luciferase CR, was found to retain its activity in various concentrations of sodium chloride. The inhibition of luciferase CR under dialysis condition was more alleviated than either Val288Ile or Glu488Val alone, suggesting that the effect of the double mutation was cumulative. We discussed the effect of mutations on the alleviation of the inhibition by sodium chloride.

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Section: Introductionmentioning

confidence: 99%

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Yawata

Noda²,

Shimomura³

et al. 2021

Biotechnol Lett

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“…Each PPI results in a higher rate of LH 2 -AMP catalysis into OxL by the Acceptor, leading to the higher light emission ( Figure 1B ). This PPI assay was named Firefly luminescent intermediate Protein-protein Interaction Assay or FlimPIA ( Ohmuro-Matsuyama et al, 2013b ; Ohmuro-Matsuyama et al, 2014 ; Kurihara et al, 2016 ; Ohmuro-Matsuyama and Ueda, 2016 , 2017 ; Ohmuro-Matsuyama et al, 2018a ; Ohmuro-Matsuyama et al, 2018b ).…”

Section: Introductionmentioning

confidence: 99%

Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase

Ohmuro‐Matsuyama

Gomi

Shimoda

et al. 2021

Front. Bioeng. Biotechnol.

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The protein–protein interaction assay is a key technology in various fields, being applicable in drug screening as well as in diagnosis and inspection, wherein the stability of assays is important. In a previous study, we developed a unique protein–protein interaction assay “FlimPIA” based on the functional complementation of mutant firefly luciferases (Fluc). The catalytic step of Fluc was divided into two half steps: D-luciferin was adenylated in the first step, while adenylated luciferin was oxidized in the second step. We constructed two mutants of Fluc from Photinus pyralis (Ppy); one mutant named Donor is defective in the second half reaction, while the other mutant named Acceptor exhibited low activity in the first half reaction. To date, Ppy has been used in the system; however, its thermostability is low. In this study, to improve the stability of the system, we applied Fluc from thermostabilized Luciola lateralis to FlimPIA. We screened suitable mutants as probes for FlimPIA and obtained Acceptor and Donor candidates. We detected the interaction of FKBP12-FRB with FlimPIA using these candidates. Furthermore, after the incubation of the probes at 37°C for 1 h, the luminescence signal of the new system was 2.4-fold higher than that of the previous system, showing significant improvement in the stability of the assay.

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“…Recently, we developed a novel PPI assay, named firefly luminescent intermediate-based proteinprotein interaction assay, FlimPIA [6][7][8][9][10]. FlimPIA utilizes the unique reaction of Fluc, which is divided into two half steps.…”

Section: Introductionmentioning

confidence: 99%

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction

Ohmuro‐Matsuyama¹,

Ueda²

2018

Protein-Protein Interaction Assays

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Protein-fragment complementation assays (PCAs) are commonly used to assay protein-protein interaction (PPI). While PCAs based on firefly luciferase (Fluc) in cells or lysates are a user-friendly method giving a high signal/background (S/B) ratio, they are difficult to use in vitro owing to the instability of split Fluc fragments. As a solution to this issue, we developed a novel protein-protein interaction assay named FlimPIA using two mutant Flucs, each of which catalyzes one of the two half-reactions catalyzed by the wild-type enzyme. Upon approximation by the tethered protein pairs, the two mutants yielded higher signal owing to a more efficient transfer of the reaction intermediate luciferyl adenylate. FlimPIA showed many advantages over in vitro split Fluc assays, such as longer detectable distance, more stable probes, and higher signal readout in a shorter time period, and it also worked in cellulo.

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A Protein–Protein Interaction Assay FlimPIA Based on the Functional Complementation of Mutant Firefly Luciferases

Cited by 3 publications

References 13 publications

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Mutant firefly luciferase enzymes resistant to the inhibition by sodium chloride

Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase

A Novel Protein-Protein Interaction Assay Based on the Functional Complementation of Mutant Firefly Luciferases: Split Structure Versus Divided Reaction

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