An enhanced chimeric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications

Abstract

Firefly luciferases, which emit visible light in a highly specific ATP-dependent process, have been adapted for a variety of applications including gene reporter assays, whole-cell biosensor measurements and in vivo imaging. We have previously reported the ~2-fold enhanced activity and 1.4-greater bioluminescence quantum yield properties of a chimeric enzyme that contains the N-domain of Photinus pyralis luciferase joined to the C-domain of Luciola italica luciferase. Subsequently, we identified 5 amino acid changes based on L. italica that are the main determinants of the improved bioluminescence properties. Further engineering to enhance thermal and pH stability produced a novel luciferase called PLG2. We present here a systematic comparison of the spectral and physical properties of the new protein with P. pyralis luciferase and demonstrate the potential of PLG2 for use in assays based on the detection of femtomol levels of ATP. Additionally, we compared the performance of a mammalian codon-optimized version of the cDNA for PLG2 with the luc2 gene in HEK293T cells. Using an optimized low-cost assay system, PLG2 activity can be monitored in mammalian cell lysates and in living cells offering an improved alternative to Promega’s luc2 for reporter and imaging applications.

Bioluminescence Imaging of Spheroids for High‐throughput Longitudinal Studies on 3D Cell Culture Models

“…Those values are consistent with those previously reported in monolayer cultures. Bioluminescence emission kinetics were measured by adding a nonlysing formulation of d ‐luciferin previously optimized for live cell imaging .…”

Imaging Adenosine Triphosphate (ATP)

“…Brachini et al have recently developed a luciferase with higher activity and quantum yield by fusing the N-terminal domain of the Photinus pyralis luciferase joined to the C-terminal domain of the Luciola italica luciferase. The chimeric luciferase was further optimized to show 3-fold higher sensitivity in live cells compared to the commercial Luc2 variant (Branchini et al , 2015). Mutagenesis has also produced luciferase variants with red-shifted luminescence (Branchini et al , 2010).…”

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

“…1A) despite its ~9-fold higher in vitro specific activity at pH 7.4 (Table 1). In these and subsequent experiments, equal numbers of living cells that expressed the human codon optimized luc genes in the pF4Ag vector were treated with 5 mM LH 2 [21]. With these standardized conditions, it was possible to make meaningful comparisons of signal intensity and stability.…”

“…Recognizing that in BLI Lucs may have to function in environments with very low ATP concentrations, we considered that the disappointing performance of PpyRE9 in HEK293T cells might be related to the K m values, and that the ATP value could be especially problematic since ATP cytoplasmic levels are ~5 fmol per viable cell [21, 22]. We therefore focused on producing a Luc variant with K m values nearer to those of CBR without seriously compromising the favorable specific activity, emission color, and thermostability of PpyRE9.…”

“…We therefore focused on producing a Luc variant with K m values nearer to those of CBR without seriously compromising the favorable specific activity, emission color, and thermostability of PpyRE9. Because PpyRE9 was so highly optimized from P. pyralis Luc, we undertook mutagenesis studies on a new template called PLG2 [21], which is a thermostable and specific activity enhanced green (λ max = 559 nm) light-emitting Luc that was engineered from a chimeric protein consisting of the large N-terminal domain of P. pyralis Luc fused to the small C-terminal domain of Luciola italica Luc. After numerous mutagenesis studies, we succeeded in transforming PLG2 into a novel Luc variant called PLR3 with the introduction of 5 amino acid changes (Supplementary Table S1).…”