Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments

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 K_m values 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 K_m value for ATP similar to CBR and ∼2.6-fold higher specific activity. The red-emitting PLR3 was ∼2.5-fold brighter than CBR in living HEK293T and HeLa cells, an improvement consistent with the importance of the K_m value in low ATP environments.

Keywords: ATP; Bioluminescence; Firefly; Imaging; Luciferase; Red-emitting.

Fig. 1

Bioluminescence activity, emission spectra, and BLI of living cells expressing luciferases. (A) Bioluminescence was initiated by the addition of 0.1 ml of 10 mM LH₂ to wells of assay plates containing equivalent numbers of live HEK293T cells expressing PLR3 (red), PpyRE9 (gray), and CBR (black) in 0.1 ml of media and monitored using a Synergy^™ 2 Multi-Mode Microplate Reader operated at 37 °C. (B) Normalized bioluminescence emission spectra of equal numbers of live HEK293T cells expressing PLR3 (red), PpyRE9 (gray), and CBR (black). The spectra were collected 1 min after the addition of 10 mM LH₂ (0.25 ml) to a cuvette containing 0.25 ml of cells (~75-fold more cells than in A) in media at 37 °C. CBR did not appear to be stable under the latter growth and assay conditions as the signal intensity was ~2.5-fold lower than expected. Additional experimental details are included in the Supplementary material. (C) BLI of live HeLa cells transfected with pF4Ag plasmids expressing luciferases. Cells (50,000) were grown in 24-well plates for 1 day. Then, 0.2 ml of 1 mM LH₂ solution in pH 5 citrate buffer was added. After 5 min, the intact live cells were imaged for 30 s with an ImagEM X2 EM-CCD camera equipped with a 10× objective and data were analyzed as described in the Supplementary material.