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Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

A luciferase from the railroad worm (Phrixothrix hirtus) is the only red‐emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site‐directed mutagenesis, we produced red‐emitting mutants with higher activity and better stability. Compared with the wild‐type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8‐fold in I212L/N351K, 8.4‐fold in I212L, and 7.8‐fold in I212L/S463R; and the cell‐based activities were 3.6‐fold in I212L/N351K and 3.4‐fold in N351K. The remaining activities after incubation at 37°C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell‐based BLI was performed, and the luminescence signal was 3.6‐fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.