Due to its small size (19kDa) and intense bioluminescence output (Figure 2), NanoLuc® luciferase is ideally suited as a protein function reporter for quantifying protein lifetime dynamics. Changes in intracellular protein levels can be quantified using Nano-Glo® reagent in a simplified assay format, wherein the luminescence output of NanoLuc® luciferase is used as a surrogate for the intracellular levels of the fusion protein. Via direct genetic fusions of NanoLuc® luciferase with HIF1A or NRF2 proteins, it is possible to measure hypoxia or oxidative stress-response signaling (respectively) in HCT116 colorectal cancer cells. Using a constitutive (e.g., CMV) promoter driving HIF1A-NanoLuc® or NRF2-NanoLuc®, changes in light output correlate to dynamic changes in protein levels. However, use of robust promoters such as CMV may result in unnaturally high intracellular levels of the protein of interest, creating potential assay artifacts.
Figure 2. A comparison of luminescence of various purified luciferases in the presence of their optimal substrates. On a molar basis, NanoLuc® luciferase is much brighter than commonly used luciferases such as firefly luciferase and Renilla luciferase.
To address this potential liability, plasmid DNA encoding genetic fusions of NanoLuc® luciferase are serially diluted into promoterless Transfection Carrier DNA to better approximate physiologically relevant expression levels of the protein of interest. To address this need, Promega offers Transfection Carrier DNA (Cat.# E4881), qualified for use in mammalian transfection experiments. In addition, a control vector, pNL3.2 [CMV], encoding NanoLuc® luciferase fused to a PEST destabilization domain (NlucP) is available as a negative control for nonspecific impacts on NanoLuc® signals. These nonspecific impacts may result from toxic chemical insults or nonspecific proteasome inhibitors introduced to cells.
As shown in Figure 3, when HCT116 cells are transiently transfected with a 1:1,000 dilution of HIF1A-NanoLuc® plasmid DNA, 1, 10-phenanthroline (a known hypoxia mimetic) induces a dose- and time-dependent accumulation of the reporter fusion. Similarly, an NRF2-NanoLuc® fusion protein accumulates in HCT116 cells upon treatment with D,L-sulforaphane (a known inducer of reactive oxygen species). To ensure proper regulation of NRF2-NanoLuc® levels in unperturbed conditions, cells are cotransfected with KEAP1 expression DNA. These results serve to demonstrate the use of NanoLuc® luciferase as a protein function reporter for monitoring regulated changes in protein stability within stress response pathways. Furthermore, these responses occurred within 2–3 hours, compared to traditional reporter gene assays that require up to 16 hours to achieve optimal responses. Protein stability reporters of HIF1A and NRF2 provide a proximal readout of the targets of these chemical stressors, enabling a highly precise mode of compound action for toxicological screens during drug discovery.
Figure 3. As a protein function reporter, NanoLuc® Luciferase enables quantitation of changes in protein stability following induction of stress response signaling. Panel A. HCT-116 cells were transiently transfected with the pNLF1-HIF1A[CMV/neo] Vector and Transfection Carrier DNA in hypoxia mimetic. Panel B. HCT-116 cells were transiently transfected with the pNLF1-NRF2[CMV/neo] Vector and pKEAP1 DNA and exposed to a stimulant. Treatments occurred as indicated and NanoLuc® Luciferase detected using Nano-Glo™ Luciferase Assay at the indicated time points using the GloMax®-Multi+ Plate Reader.
NanoLuc® luciferase also enables applications of protein stability sensors in advanced cell models. Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are an attractive cell model for studying HIF1A signaling in ischemic disease ( (2) ). As shown in Figure 4, Panel A, induction of HIF1A levels can be monitored in iCell® Cardiomyocytes transiently transfected with HIF1A-NanoLuc® fusion proteins. Prolyl hydroxylase inhibitors such as IOX2, CoCl2, ML-228, and 1, 10-phenanthroline each induced dose-dependent increases in HIF1A-NanoLuc® proteins after only 3 hours of stimulation. These results are consistent with results generated using a hypoxia response element (HRE-luc2P) firefly reporter gene assay, following 6-hour stimulation (Figure 4, Panel B). The application of the HIF1A-NanoLuc® stability sensor within iCell® Cardiomyocytes provides an elegant assay readout in a physiologically-relevant cellular context for studying HIF1A signaling.
Figure 4. A comparison of hypoxia response reporters in iPS-derived cardiomyocytes. Three to five days post seeding, iCell® Cardiomyocytes were transiently transfected using either the pNLF1-HIF1A[CMV/neo] fusion construct, diluted 1:100 into Transfection Carrier DNA (Panel A) or pGL4.42[luc2P/HRE/Hygro] HRE response element Vector (Panel B). Twenty-four hours post-transfection, cells were stimulated for either 3 hours (HIF1A-NanoLuc® fusion) or 6 hours (HRE reporter) with the indicated compounds and assayed using either ONE-Glo™ Luciferase Assay System or Nano-Glo™ Luciferase Assay Reagents, respectively. Luminescence was quantified on a GloMax®-Multi+ Plate Reader.
As a protein function reporter, NanoLuc® luciferase also provides a quantitative measure of protein levels from endogenously expressed genetic loci. As overexpression of the protein of interest may lead to assay artifacts, the ability to express the target protein at endogenous levels may be desirable. However, use of reporters with limited sensitivity provides a major challenge when the target protein is produced at endogenous levels, especially when the target protein is naturally unstable. To explore the utility of NanoLuc® luciferase as a reporter of endogenous protein stability, Horizon’s proprietary GENESIS™ platform was used to introduce the NanoLuc® gene into specific chromosomal loci as in‐frame protein fusions to HIF1A or NRF2.
Figure 5. As a protein function reporter, NanoLuc® Luciferase enables detection of proteins expressed from endogenous gene loci. Engineered HCT116 cells expressing NanoLuc® Luciferase fused to endogenous HIF1A (Panel A) or NRF2 (Panel B) were stimulated with either tBHQ or 1,10-phenanthroline, respectively, with detection performed using the Nano-Glo™ Luciferase Assay, at the indicated time points. Both fusion constructs showed a dose- and time-dependent response.
As shown in Figure 5, HCT116 cells engineered with NanoLuc® as an in-frame fusion to a single allele of HIF1A can be used to monitor inducible changes in HIF1A levels upon treatment with 1, 10 phenanthroline. HCT116 cells similarly engineered with a NRF2-NanoLuc® fusion responded in a dose- and time-dependent manner to tertbutylhydroquinone (tBHQ). These results validate the use of NanoLuc® luciferase as an ultrasensitive reporter of protein stability dynamics without the need for overexpression.
Promega has provided a suite of NanoLuc® fusion vectors to enable intracellular protein stability assays. These vectors facilitate subcloning of N-terminal and C-terminal fusions with NanoLuc® luciferase using standard restriction enzyme cloning or Promega’s convenient Flexi® cloning system (Table 2). Flexi® System-compatible vectors facilitate transfer of the gene of interest from ORF clone gene panels, as provided within the Promega “Find My Gene” offering.
For more information about NanoLuc® fusion vectors, including HIF1A-NanoLuc® and NRF2-NanoLuc® expression vectors, visit: www.promega.com/products/pm/nanoluc-redefining-reporter-assays/