Novel BRET combination for detection of rapamycin-induced protein dimerization using luciferase from fungus Neonothopanus nambi
Aaiyas Mujawar,
Shalini Dimri,
Ksenia A. Palkina,
Nadezhda M. Markina,
Karen S. Sarkisyan,
Anastasia V. Balakireva,
Ilia V. Yampolsky,
Abhijit De
Affiliations
Aaiyas Mujawar
Advanced Center for Treatment Research and Education in Cancer (ACTREC), Sector-22, Kharghar, Navi Mumbai 410210, India; Life Science, Homi Bhabha National Institute, Mumbai, India
Shalini Dimri
Advanced Center for Treatment Research and Education in Cancer (ACTREC), Sector-22, Kharghar, Navi Mumbai 410210, India; Life Science, Homi Bhabha National Institute, Mumbai, India
Ksenia A. Palkina
Planta LLC, Bolshoi Boulevard, 42 Str 1, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow, Russia
Nadezhda M. Markina
Planta LLC, Bolshoi Boulevard, 42 Str 1, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow, Russia
Karen S. Sarkisyan
Planta LLC, Bolshoi Boulevard, 42 Str 1, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow, Russia; Synthetic Biology Group, MRC London Institute of Medical Sciences, London W12 0NN, UK
Anastasia V. Balakireva
Planta LLC, Bolshoi Boulevard, 42 Str 1, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow, Russia
Ilia V. Yampolsky
Planta LLC, Bolshoi Boulevard, 42 Str 1, Moscow, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow, Russia; Corresponding author. Institute of Bioorganic Chemistry (IBCh), Russian Academy of Sciences, Moscow, Russia.
Abhijit De
Advanced Center for Treatment Research and Education in Cancer (ACTREC), Sector-22, Kharghar, Navi Mumbai 410210, India; Life Science, Homi Bhabha National Institute, Mumbai, India; Corresponding author. Molecular Functional Imaging Lab, Advanced Centre for Treatment, Research and Education in Cancer, Navi Mumbai, India.
Bioluminescence resonance energy transfer (BRET) is one of the most promising approaches used for noninvasive imaging of protein-protein interactions in vivo. Recently, our team has discovered a genetically encodable bioluminescent system from the fungus Neonothopanus nambi and identified a novel luciferase that represents an imaging tool orthogonal to other luciferin-luciferase systems. We demonstrated the possibility of using the fungal luciferase as a new BRET donor by creating fused pairs with acceptor red fluorescent proteins, of which tdTomato provided the highest BRET efficiency. Using this new BRET system, we also designed a mTOR pathway specific rapamycin biosensor by integrating the FRB and FKBP12 protein dimerization system. We demonstrated the specificity and efficacy of the new fungal luciferase-based BRET combination for application in mammalian cell culture that will provide the unique opportunity to perform multiplexed BRET assessment in the future.
