Selection and Engineering of Novel Brighter Bioluminescent Reporter Gene and Color- Tuning Luciferase for pH-Sensing in Mammalian Cells

Abstract

Firefly luciferases have been extensively used for bioanalytical applications, including their use as bioluminescent reporters, biosensors, and for bioimaging biological and pathological processes. Due to their intrinsic pH- sensitivity, in recent years we have demonstrated that firefly luciferases can also be harnessed as color- tuning sensors of intracellular pH. However, it is known that mammalian cells require temperatures higher than 36 °C, which red-shift the bioluminescence spectra of most firefly luciferases, decreasing their activities and the resolution of ratiometric pH analysis. Therefore, we prospected and engineered novel pH-sensitive firefly luciferases for mammalian cells. We humanized the luciferases of Amydetes vivianii (Amy-Luc) and Cratomorphus distinctus (Crt-Luc) fireflies, inserted them into the pCDNA3 vector, and compared their bioluminescence and pH-sensing properties with those of Macrolampis firefly luciferase (Mac-Luc) inside fibroblasts. The transfected COS-1 with Mac-Luc and Crt-Luc displayed lower bioluminescence activity and considerably red-shifted spectra (611 and 564 nm, respectively) at 37 °C, whereas Amy-Luc displayed the highest bioluminescence activity and spectral stability at 37 °C inside cells, displaying the most blue-shifted spectrum at such temperatures (548 nm) and the best spectral resolution at different pH values, making it possible to ratiometrically estimate the pH from 6.0 to 8.0. These results show that Amy-Luc is a novel brighter reporter gene and suitable pH- indicator for mammalian cells. Furthermore, whereas at pH 8.0 the spectrum was thermally stable, at pH 6.0 Amy-Luc showed higher temperature sensitivity, raising the possibility of using this luciferase as an intracellular temperature sensor. Thus, the improved bioluminescence properties as compared to existing luciferases could offer advantages for in vivo imaging and pH- sensing for the study of mammalian cellular physiology.

Keywords: bioluminescence; biosensors; intracellular events.

Figure 1

Comparison of the bioluminescent activity of four sets of COS-1 cells transfected with pCMV vector harboring the pH-sensitive luciferase genes, after the addition of D-luciferin, showing the highest intensity for cells transfected with pCMV-Amy. Photography taken with Cannon Camera (Iso: 12000; exposure time: 30 s).

Figure 2

The bioluminescent kinetic profile of COS-1 cells transfected with pH-sensitive luciferase genes for 12 h after the addition of D-luciferin, showing a higher relative intensity for cells transfected with pCMV-Amy. The error bars refer to triplicates performed for the experiment.

Figure 3

Bioluminescent COS-1 cells transfected with pCMV containing different pH-sensitive firefly luciferases at different pHs after incubation in calibration buffer containing nigericin. Cells transfected with pCMV-Amy luciferase showed the most intense bioluminescence and slight color change at different pH.

Figure 4

The effect of pH on the bioluminescence spectra of COS-1 cells transfected with plasmid pCMV containing firefly luciferases: (A) pCMV-Amy; (B) pCMV-Crt; (C) pCMV-Mac.

Figure 5

Ratiometric curves showing the effect of pH on the ratio of green and red light intensities (R = I_green/I_red) obtained from the spectra measured in the spectroluminometer: (A) pCMV-Amy (R = I₅₅₁/I₅₈₃₎; (B) pCMV-Crt (R = I₅₆₄/I₅₈₃); (C) pCMV-Mac (R = I₅₈₃/I₆₁₂). The standard deviations are shown in the Table A1.

Figure 6

The effect of pH on the bioluminescence spectra at different temperatures: (upper horizontal panel) purified Amydetes vivianii luciferase; (lower panel) COS-1 cells transfected with pCMV-Amy.

Figure 7

Ratiometric analysis of the effect of pH on green (I₅₅₀) and red (I₆₀₀) emissions (RI₅₅₀/I₆₀₀) at different temperatures: 22 °C (blue); 37 °C (orange); 45 °C (gray). The standard deviation ranged from 2 to 10%.

Figure 8

The photographic detection of bioluminescent COS-1 cells transfected with pCMV-Amy: (A) the effect of pH on the bioluminescence color of COS-1 cells transfected with pCMV-Amy at different temperatures. Photography taken with Cannon Camera (Iso: 12000; exposure time: 30 s; (B) the effect of pH on the bioluminescence color at room temperature. Photography taken with an Edge 20 pro smartphone (Motorola).