Enhanced red-emitting railroad worm luciferase for bioassays and bioimaging

Abstract

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 degrees 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.

Figure 1

Extract-based activity of WT and mutant PhRED. (A) Western blots using anti-PhRED antibody. Ctrl(−) was not transfected by expression plasmid. Tubulin was used as an internal control. (B) Bioluminescent activity normalized by quantitative analysis of western blots. The bioluminescent activity of WT was set to 1. Error bars indicate the standard deviation (n = 4).

Figure 2

Bioluminescent emission spectra of WT and mutant PhRED.

Figure 3

Cell-based activity of WT and mutant PhRED. (A) Cell-based luminescent activity in real-time monitored for about 100 h. (B) Total relative light unit during a monitoring period of about 24 h.

Figure 4

Representative bioluminescence CCD images of WT and selected mutant PhRED in NIH3T3 cells. (A) Luminescence images of WT (left panel), N351K (middle panel), and I212L/N351K (right panel). The contrast of all images was adjusted equally. (B) Luminescence intensity of WT and mutant luciferases quantified from 100 individual cells. The intensity of WT was set to 1. Error bars indicate the standard deviation.