Journal
FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY
Volume 9, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fbioe.2021.778120
Keywords
protein-protein interaction (PPI); firefly luciferase (Fluc); bioluminescence; adenylation; oxidative reaction; thermostability; assay stability
Funding
- JSPS KAKENHI from the Japan Society for the Promotion of Science, Japan [JP18H03851, JP17K06920]
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The study developed a protein-protein interaction assay, FlimPIA, based on mutant firefly luciferases, improving the stability and sensitivity of the assay system through strain modification and mutant screening.
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 degrees 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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