Red-emitting luciferases for bioluminescence reporter and imaging applications

North American firefly Photinus pyralis luciferase, which emits yellow-green light (557 nm), has been adapted for a variety of applications, including gene reporter assays, whole-cell biosensor measurements, and in vivo imaging. Luciferase variants with red-shifted bioluminescence and high specific activity can be paired with green-emitting counterparts for use in dual-color reporter assays or can be used alone for in vivo imaging. Beginning with a previously reported red-emitting thermostable mutant and using mutagenesis techniques, we engineered two luciferases with redder emission maxima while maintaining satisfactory specific activities and thermostability. The novel enzymes were expressed in HEK293 cells, where they performed similarly to Promega's codon-optimized click beetle red luciferase in model reporter assays. When the firefly luciferase variants were codon-optimized and retested using optimized substrate concentrations, they provided 50- to 100-fold greater integrated light intensities than the click beetle enzyme. These results suggest that the novel enzymes should provide superior performance in dual-color reporter and in vivo imaging applications, and they illustrate the importance of codon optimization for assays in mammalian cells.