Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Kmvalues for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Kmvalue for ATP similar to CBR and â¼2.6-fold higher specific activity. The red-emitting PLR3 was â¼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Kmvalue in low ATP environments.
Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments
Michelini, Elisa;Cevenini, Luca;Roda, Aldo;
2017
Abstract
Beetle luciferases have been adapted for live cell imaging where bioluminescence is dependent on the cellular availability of ATP, O2, and added luciferin. Previous Photinus pyralis red-emitting variants with high Kmvalues for ATP have performed disappointingly in live cells despite having much higher relative specific activities than enzymes like Click Beetle Red (CBR). We engineered a luciferase variant PLR3 having a Kmvalue for ATP similar to CBR and â¼2.6-fold higher specific activity. The red-emitting PLR3 was â¼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the Kmvalue in low ATP environments.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
