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. 2022 Sep 9;12(9):742.
doi: 10.3390/bios12090742.

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing

Maria Maddalena Calabretta  1   2 , Denise Gregucci  1   2 , Héctor Martínez-Pérez-Cejuela  1   3 , Elisa Michelini  1   2   4
Affiliations

A Luciferase Mutant with Improved Brightness and Stability for Whole-Cell Bioluminescent Biosensors and In Vitro Biosensing

Maria Maddalena Calabretta et al. Biosensors (Basel). .

Abstract

The availability of new bioluminescent proteins with tuned properties, both in terms of emission wavelength, kinetics and protein stability, is highly valuable in the bioanalytical field, with the potential to improve the sensitivity and analytical performance of the currently used methods for ATP detection, whole-cell biosensors, and viability assays among others. We present a new luciferase mutant, called BgLuc, suitable for developing whole-cell biosensors and in vitro biosensors characterized by a bioluminescence maximum of 548 nm, narrow emission bandwidth, favorable kinetic properties, and excellent pH- and thermo-stabilities at 37 and 45 °C and pH from 5.0 to 8.0. We assessed the suitability of this new luciferase for whole-cell biosensing with a cell-based bioreporter assay for Nuclear Factor-kappa B (NF-kB) signal transduction pathway using 2D and 3D human embryonic kidney (HEK293T) cells, and for ATP detection with the purified enzyme. In both cases the luciferase showed suitable for sensitive detection of the target analytes, with better or similar performance than the commercial counterparts.

Keywords: ATP; NF-kB; bioluminescence; biosensing; luciferase; whole-cell biosensors.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Locations of the mutated aminoacids, shown in green, depicted in the Ppy luciferase crystal structure (Protein Data Bank:1LCI). Modelling was performed with UCSF ChimeraX.
Figure 2
Figure 2
(a) Normalized BL emission spectrum, (b) emission kinetic and (c) heat inactivation studies of purified BgLuc luciferase mutant incubated at 37 °C obtained with Thermo Scientific Varioskan LUX Multimode Microplate Reader. Inset shows the BgLuc heat inactivation study tested until 50% activity loss.
Figure 3
Figure 3
BgLuc mutant emission spectra were obtained at different temperatures with the (a) BrightGlo and (b) D-Luciferin 1.0 mM substrates and at different pH with the (c) BrightGlo and (d) D-Luciferin 1.0 mM substrates.
Figure 4
Figure 4
Calibration curve for ATP detection obtained with BgLuc luciferase (6 μL of a 0.5 mg/mL solution). The measurements were performed in triplicate and repeated at least three times.
Figure 5
Figure 5
Dose-response curves obtained in (a) 2D or (b) 3D cell-based assays. HEK293T cells were incubated for 5 h at 37 °C with the TNFα, using BgLuc (solid line) and commercial Luc2P (dotted line) as reporters under the control of NFkB -response element. BL measurements were obtained after the addition of BrightGlo substrate as described in the Materials and Methods section. The experiment was performed in triplicate and repeated at least three times.
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