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. 2012 Nov 16;7(11):1848-57.
doi: 10.1021/cb3002478. Epub 2012 Aug 30.

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate

Mary P Hall  1 , James Unch, Brock F Binkowski, Michael P Valley, Braeden L Butler, Monika G Wood, Paul Otto, Kristopher Zimmerman, Gediminas Vidugiris, Thomas Machleidt, Matthew B Robers, Hélène A Benink, Christopher T Eggers, Michael R Slater, Poncho L Meisenheimer, Dieter H Klaubert, Frank Fan, Lance P Encell, Keith V Wood
Affiliations
Free PMC article

Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate

Mary P Hall et al. ACS Chem Biol. .
Free PMC article

Abstract

Bioluminescence methodologies have been extraordinarily useful due to their high sensitivity, broad dynamic range, and operational simplicity. These capabilities have been realized largely through incremental adaptations of native enzymes and substrates, originating from luminous organisms of diverse evolutionary lineages. We engineered both an enzyme and substrate in combination to create a novel bioluminescence system capable of more efficient light emission with superior biochemical and physical characteristics. Using a small luciferase subunit (19 kDa) from the deep sea shrimp Oplophorus gracilirostris, we have improved luminescence expression in mammalian cells ~2.5 million-fold by merging optimization of protein structure with development of a novel imidazopyrazinone substrate (furimazine). The new luciferase, NanoLuc, produces glow-type luminescence (signal half-life >2 h) with a specific activity ~150-fold greater than that of either firefly (Photinus pyralis) or Renilla luciferases similarly configured for glow-type assays. In mammalian cells, NanoLuc shows no evidence of post-translational modifications or subcellular partitioning. The enzyme exhibits high physical stability, retaining activity with incubation up to 55 °C or in culture medium for >15 h at 37 °C. As a genetic reporter, NanoLuc may be configured for high sensitivity or for response dynamics by appending a degradation sequence to reduce intracellular accumulation. Appending a signal sequence allows NanoLuc to be exported to the culture medium, where reporter expression can be measured without cell lysis. Fusion onto other proteins allows luminescent assays of their metabolism or localization within cells. Reporter quantitation is achievable even at very low expression levels to facilitate more reliable coupling with endogenous cellular processes.

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Figures

Figure 1
Figure 1
Chemical structures. (a) Coelenterazine. (b) Coelenterazine imidazopyrazinone core (with numbering scheme). (c) Furimazine and presumed reaction products.
Figure 2
Figure 2
(a) Furimazine and coelenterazine titrations using Nluc for determining relative signal intensities and Km (n = 3). Note the left and right axes have different scales. (b) Comparison of luminescence intensity (at 10 min) for purified Nluc, Fluc, and Rluc. (c) Spectral profiles for Nluc (furimazine), Rluc (coelenterazine), Fluc (d-luciferin), and click beetle red luciferase (CBR) (d-luciferin). Emission peaks: Nluc (460 nm), Rluc (480 nm), Fluc (565 nm), and CBR (605 nm). RLU = relative luminescence units.
Figure 3
Figure 3
Comparison between purified Nluc and Fluc for sensitivity to (a) elevated temperature (n = 4), (b) pH (n = 3), (c) urea (n = 3), and (d) NaCl (n = 3).
Figure 4
Figure 4
Intracellular distribution of Nluc determined by (a) confocal imaging/ICC of transient expression in U2OS cells fixed and processed with anti-Nluc IgG/Alexa488-conjugated secondary IgG (left panel = fluorescence; right panel = DIC); scale bar = 20 μm. (b) BLI of transient expression in U2OS cells; scale bar = 40 μm. (c) BLI of stable expression in Hela cells; scale bar = 40 μm. BLI was performed on an Olympus LV200 Bioluminescence Imager using a single addition of furimazine.
Figure 5
Figure 5
(a) Reporter induction by tandem cAMP response elements (CRE). Nluc, Fluc, NlucP, and FlucP were transiently expressed in HEK293 cells under multiple CRE linked to a minimal promoter; luminescence measured 5 h after adding varying concentrations of FSK (n = 3). (b) Intracellular lifetime of reporters following treatment with cycloheximide. Remaining luminescence was monitored over time (n = 3) for Nluc, NlucP, Fluc, and FlucP transiently expressed in HEK293 under a constitutive promoter. (c) Reporter induction by tandem NFκB-response elements. Nluc, Fluc, NlucP, and FlucP were transiently expressed in HEK293 cells under multiple response elements linked to a minimal promoter; fold induction determined after adding recombinant, human TNFα (100 ng/mL) by comparison of treated relative to untreated samples for each time point (n = 3). (d) Assay of reporter secreted to the culture medium. HEK293 cells transiently expressing secNluc under tandem CRE were treated with FSK (10 μM) or vehicle alone; luminescence measured periodically from aliquots of culture medium (n = 3).
Figure 6
Figure 6
Use of Nluc for monitoring regulated changes in p53 stability. HEK293 cells transiently expressing p53-Nluc or Nluc were treated with etoposide for 6 h (n = 5). Response was calculated by comparing treated samples to untreated controls.
Figure 7
Figure 7
Monitoring translocation of Nluc fusion proteins using BLI. Hela cells transiently expressing Nluc-GR fusions show (a) cytosolic localization and (b) nuclear accumulation after 15 min of dexamethasone (500 nM) treatment. U2OS cells transiently expressing Nluc-PKCα fusions show (c) cytosolic localization and (d) plasma membrane accumulation after 20 min of PMA (100 nM) treatment. Scale bar = 40 μm.
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