Abstract
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.
Highlights
The protein–protein interaction (PPI) assay is a key technology, in basic biology and for practical purposes in drug screening (Gul and Hadian, 2014; Xu et al, 2016; Ashkenazi et al, 2017), diagnosis (Yuan et al, 2017; Atan et al, 2018), and food inspection (Rasooly, 2001; Katam et al, 2016)
We previously developed a PPI assay based on the two divided catalytic reactions of firefly luciferase (Fluc); the first catalytic half-reaction involves the adenylation of D-luciferin (LH2), which produces the reaction intermediate, luciferyl adenylate (LH2-AMP), while the second half-reaction involves the oxidation of LH2-AMP, which produces oxyluciferin (OxL)
Fluc consists of N- and C-terminal domains, and the C-terminal domain rotates ∼140° according to the reaction proceeds from the first to the second half-reaction (Branchini et al, 2011; Sundlov et al, 2012)
Summary
The protein–protein interaction (PPI) assay is a key technology, in basic biology and for practical purposes in drug screening (Gul and Hadian, 2014; Xu et al, 2016; Ashkenazi et al, 2017), diagnosis (Yuan et al, 2017; Atan et al, 2018), and food inspection (Rasooly, 2001; Katam et al, 2016). Each PPI results in a higher rate of LH2-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; OhmuroMatsuyama et al, 2014; Kurihara et al, 2016; OhmuroMatsuyama and Ueda, 2016, 2017; Ohmuro-Matsuyama et al, 2018a; Ohmuro-Matsuyama et al, 2018b). The thermostability of FlimPIA remains insufficient owing to the low stability of Ppy. Fluc is applied to a hygiene monitoring system (Corbitt et al, 2000; Bakke and Suzuki, 2018), incubation of the enzyme for 1 h at 37°C resulted in an 83% decrease in signal intensity. LlL was obtained by random mutagenesis and screening of a high activity mutant L344A, encoding two mutations V287A and V392I
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