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.
Branchini, B.R., Southworth, T.L., Fontaine, D.M., Kohrt, D., Welcome, F.S., Florentine, C.M., et al. (2017). Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments. ANALYTICAL BIOCHEMISTRY, 534, 36-39 [10.1016/j.ab.2017.07.001].
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.
