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

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 fu...

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Autores principales:	Yuki Ohmuro-Matsuyama, Keiko Gomi, Takuya Shimoda, Hideki Yamaji, Hiroshi Ueda
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	protein-protein interaction (PPI) firefly luciferase (Fluc) bioluminescence adenylation oxidative reaction thermostability Biotechnology TP248.13-248.65
Acceso en línea:	https://doaj.org/article/caff5f34cadc4ec0998902aa49c54a3f
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spelling	oai:doaj.org-article:caff5f34cadc4ec0998902aa49c54a3f2021-11-11T05:43:23ZImproving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase2296-418510.3389/fbioe.2021.778120https://doaj.org/article/caff5f34cadc4ec0998902aa49c54a3f2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fbioe.2021.778120/fullhttps://doaj.org/toc/2296-4185The 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.Yuki Ohmuro-MatsuyamaYuki Ohmuro-MatsuyamaYuki Ohmuro-MatsuyamaKeiko GomiTakuya ShimodaHideki YamajiHiroshi UedaFrontiers Media S.A.articleprotein-protein interaction (PPI)firefly luciferase (Fluc)bioluminescenceadenylationoxidative reactionthermostabilityBiotechnologyTP248.13-248.65ENFrontiers in Bioengineering and Biotechnology, Vol 9 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	protein-protein interaction (PPI) firefly luciferase (Fluc) bioluminescence adenylation oxidative reaction thermostability Biotechnology TP248.13-248.65
spellingShingle	protein-protein interaction (PPI) firefly luciferase (Fluc) bioluminescence adenylation oxidative reaction thermostability Biotechnology TP248.13-248.65 Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Keiko Gomi Takuya Shimoda Hideki Yamaji Hiroshi Ueda Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
description	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.
format	article
author	Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Keiko Gomi Takuya Shimoda Hideki Yamaji Hiroshi Ueda
author_facet	Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Yuki Ohmuro-Matsuyama Keiko Gomi Takuya Shimoda Hideki Yamaji Hiroshi Ueda
author_sort	Yuki Ohmuro-Matsuyama
title	Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
title_short	Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
title_full	Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
title_fullStr	Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
title_full_unstemmed	Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase
title_sort	improving the stability of protein–protein interaction assay flimpia using a thermostabilized firefly luciferase
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/caff5f34cadc4ec0998902aa49c54a3f
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Improving the Stability of Protein–Protein Interaction Assay FlimPIA Using a Thermostabilized Firefly Luciferase

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