ACS Publications. Most Trusted. Most Cited. Most Read
Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
More

OR SEARCH CITATIONS

My Activity
Publications

Figure 1Loading Img
Download Hi-Res ImageDownload to MS-PowerPointCite This:ACS Chem. Biol. 2012, 7, 11, 1848-1857
  • Open Access
Articles

Engineered Luciferase Reporter from a Deep Sea Shrimp Utilizing a Novel Imidazopyrazinone Substrate
Click to copy article linkArticle link copied!

View Author Information
† Promega Corporation, Madison, Wisconsin 53711 United States
‡ Promega Biosciences Incorporated, San Luis Obispo, California 93401 United States
*E-mail: [email protected]
Open PDFSupporting Information (3)

ACS Chemical Biology

Cite this: ACS Chem. Biol. 2012, 7, 11, 1848–1857
Click to copy citationCitation copied!
https://doi.org/10.1021/cb3002478
Published August 15, 2012

Copyright © 2012 American Chemical Society. This publication is licensed under these Terms of Use.

Request reuse permissions

Abstract

Click to copy section linkSection link copied!
High Resolution Image
Download MS PowerPoint Slide

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.

Copyright © 2012 American Chemical Society

Subjects

what are subjects

Bioluminescence is found across a diversity of life that includes bacteria, insects, fungi, and an abundance of marine organisms. (1) It occurs when a photon-emitting substrate (luciferin) is oxidized by a generic class of enzymes called luciferases. These enzymes have been popular as reporters of cellular physiology because of their ability to provide highly sensitive quantitation with broad linearity. Firefly (Fluc, 61 kDa) and Renilla (Rluc, 36 kDa) luciferases have accounted for the majority of such applications, particularly for elucidating molecular processes coupled to gene expression. More recently, bioluminescence has been applied to other aspects of cellular analysis. Fluc has been configured into assay reagents for quantitation of cell viability, apoptosis, and various processes linked to cellular metabolism. (2, 3) Luciferases have been fused to other proteins to monitor their metabolism (4) and interactions, (5) circularly permuted to create intracellular biosensors, (6) and split into fragments to monitor protein interactions in living cells. (7)
The widely recognized utility of bioluminescence has spurred investigation of alternative luciferases, predominantly from marine organisms. Luciferase genes have been derived from the copepods Gaussia princeps (20 kDa) (8) and Metridia longa (24 kDa), (9) the ostracod Cypridina noctiluca (61 kDa), (10) the dinoflagellate Pyrocystis lunula (40 kDa), (11) and the deep sea shrimp, Oplophorus gracilirostris (106 kDa). (12)Gaussia luciferase in particular has been used as a secreted reporter in mammalian cells, (13) reportedly providing increased assay sensitivity owing to its bright luminescence and accumulation in the cell culture medium. (8) However, the light intensity decays rapidly under most conditions, thus requiring luminometers equipped with injectors to measure the transient peak luminescence. Furthermore, the coelenterazine substrate is prone to chemical instability and high autoluminescence background, (14) properties that make sample handling difficult and decrease assay sensitivity.
Although bright luminescence is generally desirable, a sustained signal with low background is necessary to enable efficient assay methods with high sensitivity. Preferably the luciferase should be small, monomeric, and structurally stable to environmental conditions. The luciferase from the deep sea shrimp, Oplophorus, suggested a route for achieving these capabilities. (15) The native luciferase is secreted by the shrimp in brilliant luminous clouds as a defense mechanism against predation. Like many marine luciferases, it utilizes coelenterazine in an ATP-independent reaction to produce blue light (spectral maximum 454 nm). The native enzyme was found to be structurally stable with a high specific activity and quantum yield. (15) Although it has a heteromeric structure consisting of two 35 kDa subunits and two 19 kDa subunits, cDNA clones revealed that bioluminescence activity was associated only with the smaller subunit (Oluc-19). (12) Unfortunately, this smaller recombinant subunit does not retain many of the desirable features evident in the native enzyme, as it is unstable and poorly expressed in the absence of the 35 kDa partner. (12)
Our examination of the Oluc-19 amino acid sequence revealed an association with the family of intracellular lipid binding proteins (iLBPs), indicating that the underlying protein architecture should support development of a stable structure. A program to achieve a more optimal enzyme structure was undertaken, which also afforded an opportunity to explore variants of the luminogenic substrate. This resulted in a novel bioluminescent system consisting of an engineered luciferase called NanoLuc (Nluc) coupled with a novel coelenterazine analogue called furimazine. The combination of these generates much brighter luminescence than Fluc or Rluc, provides improved physical and chemical characteristics, and is generally compatible with mammalian cells. Overall, we found that Nluc performed exceptionally well as a reporter and anticipate that it will further advance the use of bioluminescence for cellular analysis.

Results and Discussion

Click to copy section linkSection link copied!

Prediction of Stabilizing Amino Acids

There is no experimentally determined structure for Oluc-19, and standard sequence search methods failed to uncover significant similarities with known proteins. (12) Using fold-recognition, (16) we identified remote similarities between Oluc-19 and the well-characterized family of iLBPs. This protein family exhibits a strongly conserved structural motif, where an Arg or Lys of strand 10 hydrogen bonds with strand 1 and packs against a conserved Trp residue. (17) This stabilizing interaction is only partially retained in Oluc-19, where the conserved Arg/Lys is replaced by Asn (Supplementary Figure s1). We hypothesized that a more stable variant of Oluc-19 could be created by mutating Asn to either Arg or Lys. Both substitutions produced higher enzyme stability and improved luminescence output in bacterial lysates, further substantiating the structural similarity to iLBPs. The Arg variant (Oluc-N166R) was most improved (∼50% increased stability at 37 °C and ∼3-fold higher luminescence intensity) and was used as the template for directed evolution.

Structural Optimization and Screening of Novel Coelenterazine Substrates

The luminescence expression of Oluc-N166R was optimized in E. coli in three phases. The first phase utilized a single round of random mutagenesis and screening in bacterial lysates with coelenterazine for brighter luminescence. Eight beneficial mutations (A4E, Q11R, A33K, V44I, A54F, P115E, Q124K, and Y138I) were combined to produce the variant C1A4E. When analyzed in HEK293 cell lysates, C1A4E was approximately 29,000-fold brighter than Oluc-N166R (Table 1). Western blot analysis indicated C1A4E was produced more efficiently than Oluc-N166R in cells (Supplementary Figure s2), accounting for much of the increased luminescence. The increased expression is consistent with improved enzyme stability of C1A4E at 37 °C, where the half-life of activity retention was increased 65-fold over that of Oluc-N166R (Table 2). Despite the increased stability, gel permeation chromatography revealed that the purified protein was largely aggregated (Supplementary Figure s3).
Table 1. Luminescence in HEK293 Lysates
luciferasecoelenterazine signal intensitya,b,cfurimazine signal intensitya,b,d
Oluc-N166R0.000089 ± 0.000007(3)0.0023 ± 0.0002(78)
C1A4E2.6 ± 0.2(88,000)16 ± 1(540,000)
Nluc2.4 ± 0.3(81,000)75 ± 9(2,500,000)
Rluc0.51 ± 0.02(17,000)0.00045 ± 0.00003(15)
a

N = 4.

b

Normalized to Fluc/ONE-Glo.

c

10 μM coelenterazine.

d

50 μM furimazine. Values normalized to Oluc-19/coelenterazine are shown in parentheses.

Table 2. Signal and Enzyme Stability in HEK293 Lysates
luciferaseenzyme stabilitya,b
(37 °C; t1/2, min)		signal durationa,b
(22 °C; t1/2, min)
Oluc-N166R5.1 ± 0.4NDc
C1A4E330 ± 17(5.5 h)92 ± 5
Nluc11,000 ± 220(7.7 days)160 ± 18
Rluc99 ± 2(1.7 h)86 ± 5
Fluc7.3 ± 0.362 ± 5
a

N = 4.

b

Oluc-N166R, C1A4E, and Nluc measured using assay buffer/50 μM furimazine; Rluc measured using Renilla-Glo buffer/10 μM coelenterazine; Fluc measured using ONE-Glo.

c

ND = not determined.

The second phase of optimization focused on identification of a superior substrate and screening for further increases in luminescence. Twenty-four novel coelenterazine analogues were synthesized (Supplementary Figure s4) containing different motifs at positions 2, 6, and 8 of the imidazopyrazinone core (Figure 1). A preferred substrate would yield brighter luminescence while also having greater chemical stability and lower background autoluminescence. We anticipated that efficient catalytic utilization of a novel substrate may require corresponding modifications to the enzyme structure. Thus, together with native coelenterazine, 11 representative analogues were used to screen a random library of C1A4E mutants for brighter luminescence. Variants exhibiting brighter luminescence were then screened again with the entire panel of compounds.

Figure 1

Figure 1. Chemical structures. (a) Coelenterazine. (b) Coelenterazine imidazopyrazinone core (with numbering scheme). (c) Furimazine and presumed reaction products.

High Resolution Image
Download MS PowerPoint Slide
Although some of the library mutants revealed improvements specific to particular substrate analogues, many exhibited increased luminescence across multiple substrates. These mutations (Q18L, F54I, F68Y, L72Q, M75K, and I90V) were apparently generally stabilizing to the enzyme structure, and their combination further enhanced enzyme stability (∼10-fold over C1A4E at 37 °C) and luminescence expression. The substrate producing the brightest luminescence signal with this enzyme variant (∼25-fold over coelenterazine) was 2-furanylmethyl-deoxy-coelenterazine (furimazine; Figure 1). Furimazine was found to be more stable in cell culture media and produce lower autoluminescence (Supplementary Figure s5a,b) than coelenterazine or coelenterazine h (R2 = benzyl; Figure 1b).
The final phase of optimization focused on maximizing luminescence with the furimazine substrate. Furimazine was used to screen another random library for brighter luminescence. Beneficial mutations were identified (L27V, K33N, K43R, and Y68D) and combined to produce NanoLuc (Nluc). In total, 16 amino acid substitutions were identified over the wild-type Oluc-19, constituting alteration to about 10% of the amino acid sequence (Supplementary Figure s6). An amended gene sequence encoding Nluc was designed by optimizing codon usage for expression in mammalian cells (www.kazusa.or.jp/codon), removing potentially strong mRNA secondary structure (http://mfold.rna.albany.edu), and removing consensus promoter sequences, other transcription factor binding sites, and potential eukaryotic mRNA splice sites.
Enzymological and physical attributes resulting from this development process are summarized in Tables 1 and 2 relative to Fluc and Rluc. Although several variants of Fluc and Rluc have been described, (18-20) we chose forms that have been routinely used as benchmarks in comparative studies. Comparisons to these reporters were done using glow-type assay formats, which are generally preferred for achieving reproducible measurements across multiple samples.
Nluc paired with furimazine produced 2.5 million-fold brighter luminescence in mammalian cells relative to Oluc-19 with coelenterazine (Table 1). Because light intensity is typically correlated inversely with signal duration, these assay characteristics generally should be considered together. The luminescence produced by Nluc decayed with a half-life >2 h, significantly longer than for C1A4E. This is also longer than in the glow-type assays used for Fluc and Rluc, yet Nluc produced 75- and 89-fold more luminescence in mammalian cells, respectively.
Light intensity produced by Rluc was affected only modestly by increasing the substrate concentrations to 50 μM (Km = 15 μM for both coelenterazine and coelenterazine h). Increased coelenterazine raised the light intensity by 1.6-fold; however, the signal decayed twice as fast (Supplementary Figure s7a,b). Coelenterazine h was less efficient, raising light intensity by 1.3-fold but with similarly rapid signal decay (Supplementary Figure s7a,b).
The dramatic improvement in light output for Nluc was due in part to the change of substrate, as Nluc, C1A4E, and Oluc-N166R all produced more luminescence with furimazine compared to coelenterazine. Nluc in particular was ∼30-fold brighter with furimazine, although for Rluc it produced >1,000-fold less luminescence relative to that of coelenterazine. Nonetheless, the increased luminescence was gained mostly through improvements in protein stability, where Nluc showed markedly greater retention of activity in lysates following incubation at 37 °C (Table 2). Moreover, in contrast to C1A4E, purified Nluc appeared only as a monomer by gel permeation chromatography (Supplementary Figure s8).

NanoLuc Characterization and Comparison to Fluc and Rluc

The assay conditions for Nluc (see Methods for buffer composition) were optimized for high luminescence intensity, sustained signal duration, and good working stability under typical laboratory conditions. The reagent is added in equal volume directly to cells in culture medium to elicit steady-state luminescence with a half-life routinely >2 h. Upon combining furimazine with the assay buffer, the working solution at ambient temperature loses potency with a half-life of ∼2 days.
The apparent Km for purified Nluc using either furimazine or coelenterazine was ∼10 μM (Figure 2a), while the maximum luminescence (i.e., apparent Vmax) was ∼30-fold higher for furimazine than for native coelenterazine. This difference in brightness is consistent with the data from lysates of cells expressing Nluc (Table 1). Purified Nluc produced ∼150-fold more luminescence than either Fluc or Rluc on a per mole basis under these glow-type assay conditions (Figure 2b). Quantum yield alone cannot account for this increased luminescence, as the values for Rluc (0.05–0.1) (21, 18) and Fluc (0.4) (22) are already relatively high. Accordingly, the higher light intensity of Nluc is more likely caused by increased catalytic turnover. Strong linearity was evident for all of the luciferases, although non-linearity can occur for Nluc at higher concentrations because the higher turnover rate can deplete available substrate. However, this is a minor practical impediment since it occurs near the saturation limit of common laboratory luminometers.

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.

High Resolution Image
Download MS PowerPoint Slide
The spectral profile of Nluc revealed an emission maximum of 460 nm, consistent with the reported spectrum of the wild-type luciferase. (15) The spectrum is 20 nm blue-shifted relative to that of Rluc and about 20% narrower (Figure 2c). As a consequence, Nluc should be well suited for applications involving multiplexing with longer wavelength luminescence reporters, (23) providing dual luciferase assays with well separated spectra to support greater composite dynamic range and sensitivity. It may be particularly beneficial to pair Nluc with a beetle luciferase having an emission maximum greater than 600 nm. (24, 20) Furthermore, given the widespread use of Rluc in bioluminescence resonance energy transfer (BRET), Nluc may be preferable due to its brighter luminescence and narrow spectrum. In contrast, the blue-shifted emission may hinder usage of Nluc in animal models, where shorter wavelengths do not readily penetrate mammalian tissues.
The enzymatic activity of Nluc was found to be more robust than that of Fluc when compared under a variety of environmental conditions. Nluc showed greater thermal stability, retaining activity following incubation at 55 °C for 30 min, whereas Fluc began losing activity below 30 °C (Figure 3a). Using buffers ranging from pH 5 to 9 (Figure 3b), Nluc displayed a broader optimal range between pH 7 and 9 and retained significant activity under more acidic conditions. In contrast, Fluc showed a narrower pH profile with activity dropping sharply below pH 8. Urea sensitivity (Figure 3c) was compared by treatment for 30 min followed by 20,000-fold dilution into assay buffer containing 50 μM furimazine for Nluc or into ONE-Glo for Fluc. Nluc maintained its activity following exposure up to 8 M, whereas Fluc lost activity above 2 M. Both Nluc and Fluc tolerated high concentrations of NaCl, but Nluc showed less inhibition at 10 M (Figure 3d).

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).

High Resolution Image
Download MS PowerPoint Slide

Performance as a Genetic Reporter

In addition to producing bright and robust luminescence, it is important that reporters be expressed without bias in their experimental hosts. By originating from a marine invertebrate and having modifications in both the enzyme and gene, Nluc is unlikely to exhibit biases unique to mammalian cells. Nonetheless, the possibility of spurious interactions was examined. Immunodetection (Figure 4a) and luminescence imaging (Figure 4b,c) both suggested that Nluc was expressed uniformly in mammalian cells, including the nuclei. Moreover, no morphological differences were evident between cells expressing Nluc and control cells (not shown). Western blots prepared from cells expressing Nluc revealed only a single band of the expected molecular weight (Supplementary Figures s2, s11). Total mass analysis by mass spectrometry (LC–MS), using purified enzyme from E. coli and mammalian cells, indicated the absence of post-translational modifications. The proteins from both sources revealed identical molecular masses, matching the calculated mass for the expected unmodified protein.

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.

High Resolution Image
Download MS PowerPoint Slide
The intracellular lifetime of a genetic reporter can substantially shape its expression characteristics. A stable reporter can persist longer in cells and thus accumulate to greater levels, allowing greater assay sensitivity and reduced variability from aberrant fluctuations in gene expression. However, by this same consideration, a stable reporter has diminished ability to detect changes in transcriptional rate. Reduced lifetime will yield less signal but may provide better response dynamics. (25) Which scenario is appropriate depends on the experimental objectives.
To achieve better response dynamics, Fluc having an appended 41-amino-acid PEST sequence is often used to shorten its intracellular lifetime. (25) We evaluated this approach by making a similar fusion to Nluc (NlucP; Supplementary Figure s6) and inserted both Nluc and NlucP into expression plasmids containing tandem cAMP-response elements (CRE). These plasmids, along with analogous plasmids containing Fluc or FlucP, were introduced into mammalian cells, and gene expression was induced by titrated addition of forskolin (FSK). FSK activates adenylate cyclase, causing an increase of intracellular cAMP to stimulate the CRE sites. All four reporters yielded similar response profiles and showed identical EC50 values (Figure 5a). As expected, NlucP was dimmer than Nluc but showed a faster rate of signal increase and greater overall response (2,000-fold versus 750-fold) following pathway activation. When compared to their Fluc counterparts over a range of similar experiments, Nluc was generally brighter than Fluc (on average 80-fold), and NlucP was brighter than FlucP (on average 10-fold).

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).

High Resolution Image
Download MS PowerPoint Slide
It was surprising to find that the PEST sequence had a larger effect on brightness and response dynamics when combined with Nluc rather than Fluc. We estimated intracellular lifetime by adding cycloheximide to block protein synthesis and measuring the decline of residual activity in the cells (Figure 5b). Both Nluc and Fluc were stable for at least 6 h. From multiple experiments, Nluc appears to have a longer lifetime than Fluc, which is consistent with the increased physical stability of Nluc. However, quantitative assessments by this method are difficult for half-lives beyond 6 h due to the toxicity of cycloheximide. As expected, FlucP had a shorter half-life (2 h) due to degradation induced by the PEST sequence. Yet, despite the high physical stability of Nluc, the intracellular half-life of NlucP (20 min) was 6-fold shorter than for FlucP.
It can be expected, therefore, that NlucP may provide better coupling to transcriptional dynamics while still providing good assay sensitivity. The influence of the intracellular lifetime is evident by the relative response of different reporters coupled to an NFκB-response element (Figure 5c). Upon stimulation with TNFα, the least relative response was achieved by Nluc, although it produced the most luminescence. The greatest relative response was achieved by NlucP, where the signal was produced faster and with higher signal over background. Both Fluc and FlucP produced intermediate responses. The greater responsiveness of NlucP as a reporter may be particularly important when the underlying genetic response of interest is subtle (Supplementary Figure s9).
Transcriptional reporters are commonly used for the high-throughput screening (HTS) of diverse compound libraries, where Fluc in particular has gained widespread use. However, the influence of chemical compounds on reporter activity, such as by inhibition or stabilization, can lead to false hits in the screening results. (26) The performance of Nluc in the context of a diverse chemical collection was assessed by screening purified enzyme against the LOPAC1280 library (Supplementary Figure s10a). Relative to a parallel screen using Fluc (Supplementary Figure s10b), the data for Nluc showed a tighter distribution with lower inhibitor potency. As a percentage of total activity, 1.2% of the library compounds inhibited Nluc by >10%, 0.5% of these inhibited by >20%, and no compounds inhibited by >30%. For Fluc, 1.9% of compounds inhibited by >10%, where 0.8% inhibited by >20% and 0.5% inhibited by >30%. We speculate that greater structural rigidity associated with the increased thermal stability of Nluc may reduce its potential to bind nonspecifically to small molecules.
The secretion of bioluminescence by the Oplophorus shrimp suggested that Nluc might retain the capacity for efficient secretion from mammalian cells. Like other secreted proteins, the Oplophorus luciferase contains multiple cysteine residues, which are thought generally to provide stability in extracellular environments through disulfide bonds. (27) For instance, Gaussia luciferase is a secreted enzyme that contains 11 cysteines. These cysteines all contribute to enzymatic activity but can hinder expression of functional enzyme within the reducing environment of the cell interior. (28) Although the Oplophorus luciferase contains 12 cysteines, all but one are located in the larger 35 kDa subunit. (12) Nluc, derived from the smaller subunit, contains only one non-essential (29) cysteine and thus has no disulfide bonds. Hence, it is suited for both intracellular and secreted reporter configurations.
Initial reports of the recombinant Oluc-19 suggested that the signal sequence may not work effectively in mammalian cells. (12) Thus we evaluated the capacity for secretion by appending both the native Oluc-19 secretion peptide and the secretion signal from human IL6 (30) to the N-terminus of Nluc. Although both peptides caused Nluc to accumulate in the media, the IL6 construct (secNluc; Supplementary Figure s6) produced a brighter signal when transiently expressed in HEK293 and Hela cells, with ∼99% of the total luminescence localized to the cell culture medium. The half-life of secreted Nluc in the culture medium at 37 °C was 4.2 ± 0.2 days for HEK293 cells (DMEM + 10% FBS) and 7.2 ± 0.3 days for CHO cells (F12 + 10% FBS). Thus, secreted reporter activity can be retained in culture conditions with negligible loss (<10%) for over 15 h. These values are comparable to what has been observed for secreted Gaussia luciferase. (13)
Western blot analysis indicated the presence of processed secNluc in the medium, while Nluc without a secretion sequence was detected only in cell lysates (Supplementary Figure s11). The blot also indicates a similar size for secNluc and Nluc in cell lysates, consistent with simultaneous translation and signal peptide processing of secNluc. Images of U2OS cells by immunocytochemistry show a punctate staining pattern consistent with vesicular transport through the secretory pathway (31) (Supplementary Figure s12). The performance of secNluc as a transcriptional reporter was evaluated using a plasmid as before containing multiple CRE sites. Following a medium change to remove secNluc activity resulting from basal expression, cells were treated with FSK to observe both the kinetics of signal accumulation (Figure 5d) and a dose response (Supplementary Figure s13). As expected, FSK caused a steady accumulation of secNluc in the medium at levels significantly higher than cells treated with vehicle alone.

Performance as a Fusion Reporter

The enhanced luminescence and small size of Nluc make it an attractive candidate as a protein fusion tag for a variety of applications. (32) Luciferase fusions have become increasingly popular for measuring regulated changes in intracellular protein lifetime via the ubiquitin/proteasome system (UPS). (33, 4) For instance, such fusions have been used to monitor stress response pathways, where the activity of a transcription factor is typically regulated by altering intracellular abundance. (34) We applied this approach by fusing Nluc to the C-terminus of p53, which is known to degrade rapidly in unstressed cells. However, DNA damaging agents (e.g., etoposide) cause the transcription factor to accumulate within cells by decoupling this degradation. Upon treatment with etoposide we observed a dose-dependent increase in signal (15-fold response) for cells expressing the p53 fusion, while no effect was evident for cells expressing Nluc alone (Figure 6). This result indicates the potential for Nluc as a fusion tag for monitoring intracellular protein lifetimes as indicators of cellular stress.

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.

High Resolution Image
Download MS PowerPoint Slide
We also examined the ability to monitor subcellular localization by bioluminescence imaging (BLI) using Nluc fusions to proteins with distinct static and dynamic localization patterns. We observed that a Histone H3-Nluc fusion was appropriately localized to the nucleus (Supplementary Figure s14a–c). When fused (with a secretion signal) to the N-terminus of the β2-adrenergic receptor (β2-AR), luminescence was localized to the plasma membrane, indicating proper trafficking through the secretory pathway (Supplementary Figure s14d). BLI has the potential for revealing protein dynamics in living cells without the need for repeated sample excitation, a cytotoxic artifact associated with use of fluorescent proteins. However, the limited brightness of existing luciferase enzymes has hindered the common use of BLI for real-time measurements. Using a Nluc-glucocorticoid receptor (GR) fusion, we monitored the expected translocation from the cytosol to the nucleus upon treatment with dexamethasone (Figure 7a,b). Finally, a fusion of Nluc to protein kinase C alpha (PKCα) was properly recruited to the plasma membrane following treatment with phorbol-12-myristate-13-acetate (PMA) (Figure 7c,d).

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.

High Resolution Image
Download MS PowerPoint Slide
The behavior of the PKCα and GR fusion proteins was consistent with the subcellular localization dynamics of endogenous PKCα (35) and GR, (36) suggesting that Nluc did not significantly perturb the functionality of the fusion partner. Of significant note, the required sample exposure times in these experiments ranged from 1 to 5 s, in contrast to the 3–10 min previously reported when using an enhanced beetle luciferase adapted specifically for BLI. (37) Exposure times exceeding 1 min would make video-rate analysis of rapid (<20 min) translocation events prohibitively difficult, whereas the short exposure times used for the Nluc fusions enabled continuous monitoring of both Nluc-GR (Supporting Video 1) and Nluc-PKCα (Supporting Video 2). Gaussia luciferase has been used for high frame-rate BLI of insulin secretion; (38) however, its reduced intracellular activity may limit its general use for this type of application.
Bioluminescent images were acquired immediately following addition of 20 μM furimazine to the culture medium. No significant morphological changes were evident under these conditions (Figure 4b,c; Supplementary Figure s15a–f). Quantitation of cell viability following 2 h of exposure showed that furimazine was tolerated up to about 20 μM (Supplementary Figure s16a). Similar viability profiles were observed for both native coelenterazine and coelenterazine h. Measurement of luminescence intensity from cells expressing Nluc revealed that maximal signal was attained at about 10–20 μM furimazine (Supplementary Figure s16b). These results indicate that high light intensity can be achieved from Nluc in living cells with minimal perturbance to their normal physiology.

Summary

Bioluminescence has been associated primarily with quantifying genetic processes, although increasingly it is proving valuable to other aspects of cellular analysis. The potential for achieving new capabilities has motivated the search for new luminescent chemistries able to deliver greater sensitivity and adaptability to experimental programs. We recognized that the Oplophorus luciferase offered such an opportunity by the inherent luminescent efficiency of the native enzyme combined with the discovery that the small catalytic subunit was structurally related to iLBPs. Because these proteins exhibit well-behaved structural properties and are ubiquitously expressed in vertebrates and invertebrates, this protein scaffold provided a good candidate for development of a robust luminescent reporter. The reportedly broad substrate specificity of Oplophorus luciferase also afforded the opportunity to design an improved luminogenic substrate. (21)
To achieve this, we combined structural optimization of the small catalytic subunit using various schemes of mutagenesis with the organic synthesis of a panel of novel substrate analogues. By folding these aspects together into an integrated process, we created a small luciferase called NanoLuc (Nluc; 19 kDa) capable of producing very bright and sustained luminescence. While elevated light intensity in short bursts has been possible with other luciferases, achieving sustained luminescence at these high levels has been elusive. Sustained luminescence greatly simplifies the instrumentation and processing requirements for sample quantification, and is essential for analysis using laboratory automation.
Nluc exhibits high physical stability despite being much smaller than Fluc, showing much greater tolerance to temperature, pH, and urea. In cells, Nluc is present as a single molecular species devoid of post-translational modifications and is uniformly distributed without apparent compartmental biases. The novel substrate, furimazine, yields light intensity higher than that of native coelenterazine and is more stable with lower background autoluminescence. The combination of these characteristics positions Nluc to be generally useful as a cellular reporter, generating a highly sensitive signal with a physical constitution that is robust to environmental influences. Nluc is particularly suited for high-throughput screening, where the bright luminescence facilitates measurements in very small sample volumes and the assay is resistant to interference from library compounds.
The structure of Nluc can be readily adapted to meet different experimental needs. It may be appended with a degradation signal (e.g., PEST) to allow expression levels to change rapidly in response to transcriptional dynamics or appended with a secretion sequence to allow export into the culture medium. The stability of Nluc does not rely on having disulfide bonds, so the enzyme may be efficiently expressed either inside or outside of cells. The small size of Nluc makes it well suited as a protein fusion tag, allowing luminescence to be associated with the physiological dynamics of specific intracellular proteins. For example, changes in luminescence can be correlated to the regulated degradation associated with many transcription factors. Luminescence from Nluc can be generated within living cells, providing sufficient signal intensity for imaging the subcellular location of fusion proteins. Further investigation will determine how broad the application space is for Nluc. The properties described here are expected to enable successful use in BRET, where the enhanced brightness over Rluc should improve assay sensitivity. Nluc may also provide unique opportunities for the development of protein complementation assays, where small size, brightness, and structural stability may offer advantages over existing approaches.

Methods

Click to copy section linkSection link copied!

Synthesis of Coelenterazine Analogues

Details on the synthesis and characterization of furimazine and the other 23 analogues (Supplementary Figure s4) can be found in the Supporting Information.

Variant Enzyme Screening

Random libraries were generated by error-prone PCR (average of 2–3 mutations per clone). Library 1 (phase 1; template = Oluc-N166R) was screened (4,400 variants) with coelenterazine. Library 2 (phase 2; template = C1A4E) was screened (4,400 variants) with 11 novel coelenterazine analogues: 3840, 3841, 3842, 3857, 3880, 3881, 3886, 3887, 3889, 3897, and 3900 (Supplementary Figure s4). The 11 analogues represented substitutions at positions 2, 6, and 8 and were considered to be representative of the entire set of 24 compounds; 2,200 variants were screened with compounds 3896 and 3894 (Supplementary Figure s4). All hits (improved luminescence) were screened again with the remaining coelenterazine analogues. Library 3 (phase 3; template = C1A4E + Q18L/K33N/F54I/F68Y/L72Q/M75K/I90V) was screened in the context of a mouse Id-X-HaloTag (where X = library) using coelenterazine and furimazine (Figure 1c). Library screens were performed on a Freedom robotic workstation (Tecan) as follows: induced bacterial cultures (in 96-well microtiter plates) were lysed with a buffer containing 300 mM HEPES pH 8, 200 mM thiourea, 0.3X Passive Lysis Buffer (PLB, Promega), 0.3 mg mL–1 lysozyme, and 0.002 units of RQ1 DNase (Promega). Assay reagent containing 1 mM CDTA, 150 mM KCl, 10 mM DTT, 0.5% (v/v) Tergitol, and 20 μM substrate was then added to equal volumes of lysate. Samples were measured on a GENios Pro luminometer (Tecan). Secondary screening to confirm hits (defined as those variants producing greater luminescence compared to that of the parental clone) and to test combination sequences was completed using a similar protocol but in manual fashion and in triplicate.

NanoLuc Assay buffer

The buffer for Nluc reactions consisted of 100 mM MES pH 6.0, 1 mM CDTA, 0.5% (v/v) Tergitol, 0.05% (v/v) Mazu DF 204, 150 mM KCl, 1 mM DTT, and 35 mM thiourea. Furimazine substrate was added to give a working reagent that was then added in equal volume directly to assay samples (final concentration of furimazine in the assay was commonly between 10 and 50 μM).
Complete methods and additional details can be found in the Supporting Information.

Supporting Information

Click to copy section linkSection link copied!

This material is available free of charge via the Internet at http://pubs.acs.org

Terms & Conditions

Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.

Author Information

Click to copy section linkSection link copied!
  • Corresponding Author
    • Lance P. Encell - Promega Corporation, Madison, Wisconsin 53711 United States;  Email: [email protected]
  • Authors
    • Mary P. Hall - Promega Corporation, Madison, Wisconsin 53711 United States
    • James Unch - Promega Biosciences Incorporated, San Luis Obispo, California 93401 United States
    • Brock F. Binkowski - Promega Corporation, Madison, Wisconsin 53711 United States
    • Michael P. Valley - Promega Corporation, Madison, Wisconsin 53711 United States
    • Braeden L. Butler - Promega Corporation, Madison, Wisconsin 53711 United States
    • Monika G. Wood - Promega Corporation, Madison, Wisconsin 53711 United States
    • Paul Otto - Promega Corporation, Madison, Wisconsin 53711 United States
    • Kristopher Zimmerman - Promega Corporation, Madison, Wisconsin 53711 United States
    • Gediminas Vidugiris - Promega Corporation, Madison, Wisconsin 53711 United States
    • Thomas Machleidt - Promega Corporation, Madison, Wisconsin 53711 United States
    • Matthew B. Robers - Promega Corporation, Madison, Wisconsin 53711 United States
    • Hélène A. Benink - Promega Corporation, Madison, Wisconsin 53711 United States
    • Christopher T. Eggers - Promega Corporation, Madison, Wisconsin 53711 United States
    • Michael R. Slater - Promega Corporation, Madison, Wisconsin 53711 United States
    • Poncho L. Meisenheimer - Promega Biosciences Incorporated, San Luis Obispo, California 93401 United States
    • Dieter H. Klaubert - Promega Biosciences Incorporated, San Luis Obispo, California 93401 United States
    • Frank Fan - Promega Corporation, Madison, Wisconsin 53711 United States
    • Keith V. Wood - Promega Corporation, Madison, Wisconsin 53711 United States
  • Notes
    The authors declare no competing financial interest.

Acknowledgment

Click to copy section linkSection link copied!

We thank M. Scurria, R. Arbit, H. Wang, L. Bernad, D. Simpson, R. Hurst, S. Saveliev, A. Niles, M. O’Brien, E. Strauss, J. Wilkinson, and T. Lubben for technical expertise and insightful discussions. We also thank G. Colwell at Gene Dynamics, LLC for help with vector constructions and J. Bujnicki at the IIMCB in Warsaw, Poland for assistance with fold-recognition analysis.

References

Click to copy section linkSection link copied!

This article references 38 other publications.

  1. 1
    Widder, E. A. (2010) Bioluminescence in the ocean: origins of biological, chemical, and ecological diversity Science 328, 704– 708
    Google Scholar
    1
    Bioluminescence in the ocean: Origins of biological, chemical, and ecological diversity
    Widder, E. A.
    Science (Washington, DC, United States) (2010), 328 (5979), 704-708CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
    A review. From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chem. generation of light) for finding food, attracting mates, and evading predators. Disparate biochem. systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecol. benefits of bioluminescence, with biochem. and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, the author reviews recent advances in understanding bioluminescence in the ocean and highlights future research efforts that will unite mol. details with ecol. and evolutionary relations.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsFCitbg%253D&md5=c07748c8f13482b1ae8362cec0ffe5e1
  2. 2
    Melnick, J. S., Janes, J., Kim, S., Chang, J. Y., Sipes, D. G., Gunderson, D., Jarnes, L., Matzen, J. T., Garcia, M. E., Hood, T. L., Beigi, R., Xia, G., Harig, R. A., Asatryan, H., Yan, S. F., Zhou, Y., Gu, X. J., Saadat, A., Zhou, V., King, F. J., Shaw, C. M., Su, A. I., Downs, R., Gray, N. S., Schultz, P. G., Warmuth, M., and Caldwell, J. S. (2006) An efficient rapid system for profiling the cellular activities of molecular libraries Proc. Natl. Acad. Sci. U.S.A. 103, 3153– 3158
    Google Scholar
    There is no corresponding record for this reference.
  3. 3
    Doshi, U. and Li, A. P. (2011) Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction J. Biomol. Screen. 16, 903– 909
    Google Scholar
    3
    Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction
    Doshi, Utkarsh; Li, Albert P.
    Journal of Biomolecular Screening (2011), 16 (8), 903-909CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
    The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (max. inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metab. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (max. inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metab., whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large no. of chems. in the drug discovery phase for CYP3A4 inhibitory and inducing potential.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1yitrnM&md5=2533a5fda46939de0805661c6b61a090
  4. 4
    Smirnova, N. A., Haskew-Layton, R. E., Basso, M., Hushpulian, D. M., Payappilly, J. B., Speer, R. E., Ahn, Y. H., Rakhman, I., Cole, P. A., Pinto, J. T., Ratan, R. R., and Gazaryan, I. G. (2011) Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators Chem. Biol. 18, 752– 765
    Google Scholar
    4
    Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators
    Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.
    Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (6), 752-765CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
    Summary: The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degrdn. of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biol. active compds. to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found-nordihydroguaiaretic acid, fisetin, and gedunin-induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotVejtbc%253D&md5=3919f582cce67396e5b869ce7b9e0325
  5. 5
    Perroy, J., Pontier, S., Charest, P. G., Aubry, M., and Bouvier, M. (2004) Real-time monitoring of ubiquitination in living cells by BRET Nat. Methods 1, 203– 208
    Google Scholar
    5
    Real-time monitoring of ubiquitination in living cells by BRET
    Perroy, Julie; Pontier, Stephanie; Charest, Pascale G.; Aubry, Muriel; Bouvier, Michel
    Nature Methods (2004), 1 (3), 203-208CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiol. environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioLuminescence resonance energy transfer (BRET), we measured the ubiquitination of β-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addn. to allowing the detection of basal and GPCR-regulated ubiquitination of p-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVGhs74%253D&md5=ae20928d2b7c8208099f526431f89154
  6. 6
    Fan, F., Binkowski, B. F., Butler, B. L., Stecha, P. F., Lewis, M. K., and Wood, K. V. (2008) Novel genetically encoded biosensors using firefly luciferase ACS Chem. Biol. 3, 346– 351
    Google Scholar
    6
    Novel Genetically Encoded Biosensors Using Firefly Luciferase
    Fan, Frank; Binkowski, Brock F.; Butler, Braeden L.; Stecha, Peter F.; Lewis, Martin K.; Wood, Keith V.
    ACS Chemical Biology (2008), 3 (6), 346-351CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
    Genetically encoded biosensors have proven valuable for real-time monitoring of intracellular phenomena, particularly FRET-based sensors incorporating variants of green fluorescent protein. To increase detection sensitivity and response dynamics, the authors genetically engineered firefly luciferase to detect specific intermol. interactions through modulation of its luminescence activity. This concept has been applied in covalent, noncovalent, and allosteric design configurations. The covalent design gives sensitive detection of protease activity through a cleavage-dependent increase in luminescence. The noncovalent and allosteric designs allow reversible detection of the small mols. rapamycin and cAMP, resp. These sensors allow detection of mol. processes within living cells following addn. of the luciferin substrate to the growth medium. For example, the cAMP sensor allows monitoring of intracellular signal transduction assocd. with G-protein coupled receptor function. These and other luminescent biosensors will be useful for the sensitive detection of cellular physiol. in research and drug discovery.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXntlKrsrY%253D&md5=59474ab5133cadb6752eed96e89fe96c
  7. 7
    Remy, I. and Michnick, S. W. (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase Nat. Methods 3, 977– 979
    Google Scholar
    7
    A highly sensitive protein-protein interaction assay based on Gaussia luciferase
    Remy, Ingrid; Michnick, Stephen W.
    Nature Methods (2006), 3 (12), 977-979CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chem. reversal, and induction and inhibition of a key interaction linking insulin and TGFβ signaling.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1Wqu77I&md5=6d80a3eded2f378506dff827c5af3e04
  8. 8
    Tannous, B. A., Kim, D. E., Fernandez, J. L., Weissleder, R., and Breakefield, X. O. (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo Mol. Ther. 11, 435– 443
    Google Scholar
    8
    Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo
    Tannous, Bakhos A.; Kim, Dong-Eog; Fernandez, Juliet L.; Weissleder, Ralph; Breakefield, Xandra O.
    Molecular Therapy (2005), 11 (3), 435-443CODEN: MTOHCK; ISSN:1525-0016. (Elsevier)
    Photoproteins have played a major role in advancing our understanding of biol. processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. HGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under std. imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsVOju7w%253D&md5=bc4ba7b78ea19d52184ebf6415cc410b
  9. 9
    Markova, S. V., Golz, S., Frank, L. A., Kalthof, B., and Vysotski, E. S. (2004) Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme J. Biol. Chem. 279, 3212– 3217
    Google Scholar
    There is no corresponding record for this reference.
  10. 10
    Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., and Ohmiya, Y. (2004) cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca Biosci. Biotechnol. Biochem. 68, 565– 570
    Google Scholar
    There is no corresponding record for this reference.
  11. 11
    Suzuki, C., Nakajima, Y., Akimoto, H., Wu, C., and Ohmiya, Y. (2005) A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells Gene 344, 61– 66
    Google Scholar
    There is no corresponding record for this reference.
  12. 12
    Inouye, S., Watanabe, K., Nakamura, H., and Shimomura, O. (2000) Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase FEBS Lett. 481, 19– 25
    Google Scholar
    There is no corresponding record for this reference.
  13. 13
    Wurdinger, T., Badr, C., Pike, L., de Kleine, R., Weissleder, R., Breakefield, X. O., and Tannous, B. A. (2008) A secreted luciferase for ex vivo monitoring of in vivo processes Nat. Methods 5, 171– 173
    Google Scholar
    13
    A secreted luciferase for ex vivo monitoring of in vivo processes
    Wurdinger, Thomas; Badr, Christian; Pike, Lisa; de Kleine, Ruben; Weissleder, Ralph; Breakefield, Xandra O.; Tannous, Bakhos A.
    Nature Methods (2008), 5 (2), 171-173CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
    Luciferases are widely used to monitor biol. processes. Here we describe the naturally secreted Gaussia princeps luciferase (Gluc) as a highly sensitive reporter for quant. assessment of cells in vivo by measuring its concn. in blood. The Gluc blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in exptl. disease and therapy models.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsV2ksLw%253D&md5=e0cc4226e91256ae0e481f84ab4f3a77
  14. 14
    Andreu, N., Zelmer, A., Fletcher, T., Elkington, P. T., Ward, T. H., Ripoll, J., Parish, T., Bancroft, G. J., Schaible, U., Robertson, B. D., and Wiles, S. (2010) Optimisation of bioluminescent reporters for use with mycobacteria PLoS One 5, e10777
    Google Scholar
    There is no corresponding record for this reference.
  15. 15
    Shimomura, O., Masugi, T., Johnson, F. H., and Haneda, Y. (1978) Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilirostris Biochemistry 17, 994– 998
    Google Scholar
    There is no corresponding record for this reference.
  16. 16
    Kurowski, M. A. and Bujnicki, J. M. (2003) GeneSilico protein structure prediction meta-server Nucleic Acids Res. 31, 3305– 3307
    Google Scholar
    There is no corresponding record for this reference.
  17. 17
    Flower, D. R., North, A. C., and Sansom, C. E. (2000) The lipocalin protein family: structural and sequence overview Biochim. Biophys. Acta 1482, 9– 24
    Google Scholar
    There is no corresponding record for this reference.
  18. 18
    Loening, A. M., Fenn, T. D., Wu, A. M., and Gambhir, S. S. (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output Protein Eng., Des. Sel. 19, 391– 400
    Google Scholar
    18
    Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output
    Loening, Andreas Markus; Fenn, Timothy David; Wu, Anna M.; Gambhir, Sanjiv Sam
    Protein Engineering, Design & Selection (2006), 19 (9), 391-400CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
    Luciferases, which have seen expansive employment as reporter genes in biol. research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational anal. were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output.These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xotl2ksro%253D&md5=c006a4ff7a42c04c1ccf02b80fb1c9ff
  19. 19
    Woo, J. and von Arnim, A. G. (2008) Mutational optimization of the coelenterazine-dependent luciferase from Renilla Plant Methods 4, 23
    Google Scholar
    There is no corresponding record for this reference.
  20. 20
    Branchini, B. R., Ablamsky, D. M., Davis, A. L., Southworth, T. L., Butler, B., Fan, F., Jathoul, A. P., and Pule, M. A. (2010) Red-emitting luciferases for bioluminescence reporter and imaging applications Anal. Biochem. 396, 290– 297
    Google Scholar
    There is no corresponding record for this reference.
  21. 21
    Inouye, S. and Shimomura, O. (1997) The use of Renilla luciferase, Oplophorus luciferase, and Apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate Biochem. Biophys. Res. Commun. 233, 349– 353
    Google Scholar
    There is no corresponding record for this reference.
  22. 22
    Ando, Y., Niwa, K., Yamada, N., Enomoto, T., Irie, T., Kubota, H., Ohmiya, Y., and Akiyama, H. (2008) Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission Nat. Photonics 2, 44– 47
    Google Scholar
    22
    Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission
    Ando, Yoriko; Niwa, Kazuki; Yamada, Nobuyuki; Enomoto, Toshiteru; Irie, Tsutomu; Kubota, Hidehiro; Ohmiya, Yoshihiro; Akiyama, Hidefumi
    Nature Photonics (2008), 2 (1), 44-47CODEN: NPAHBY; ISSN:1749-4885. (Nature Publishing Group)
    Firefly bioluminescence is the most well-known ideal photoemitter system in biophotonics, known in particular for its extremely high quantum yield, 88 ± 25% (refs. 2,3) or higher, and its magnificent pH-dependent emission-color change between yellow-green and red, modelled as the chem. equil. between two corresponding states. However, the need for re-examn. has also been discussed. In this letter we quantify quantum yields and color changes using our new total-photon-flux spectrometer. We det. the highest quantum yield to be 41.0 ± 7.4% (1 std. deviation (s.d.) est., coverage factor k = 1), and find that bioluminescence spectra are systematically decompd. into one pH-sensitive and two pH-insensitive gaussian components. There is no intensity conversion between yellow-green and red emissions through pH equil., but simple intensity variation of the pH-sensitive gaussian peak at 2.2 eV causes the changes in emission colors. This represents a paradigm shift in the concept of color detn. from long-standing interpretation based on pH equil.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmtVSnuw%253D%253D&md5=00549500c02c8644684b672d7530e863
  23. 23
    Davis, R. E., Zhang, Y. Q., Southall, N., Staudt, L. M., Austin, C. P., Inglese, J., and Auld, D. S. (2007) A cell-based assay for IκBα stabilization using a two-color dual luciferase-based sensor Assay Drug Dev. Technol. 5, 85– 103
    Google Scholar
    There is no corresponding record for this reference.
  24. 24
    Almond, B., Hawkins, E., Stecha, P., Garvin, D., Paguio, A., Butler, B. L., Beck, M., Wood, M., and Wood, K. (2003) Introducing ChromaLuc technology Promega Notes 85, 11– 14
    Google Scholar
    There is no corresponding record for this reference.
  25. 25
    Swanson, B., Fan, F., and Wood, K. V. (2007) Enhanced response dynamics for transcription analysis using new pGL4 luciferase reporter vectors Cell Notes 17, 3– 5
    Google Scholar
    There is no corresponding record for this reference.
  26. 26
    Auld, D. S., Zhang, Y. Q., Southall, N. T., Rai, G., Landsman, M., MacLure, J., Langevin, D., Thomas, C. J., Austin, C. P., and Inglese, J. (2009) A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution J. Med. Chem. 52, 1450– 1458
    Google Scholar
    26
    A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution
    Auld, Douglas S.; Zhang, Ya-Qin; Southall, Noel T.; Rai, Ganesha; Landsman, Marc; MacLure, Jennifer; Langevin, Daniel; Thomas, Craig J.; Austin, Christopher P.; Inglese, James
    Journal of Medicinal Chemistry (2009), 52 (5), 1450-1458CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
    We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quant. high-throughput screening (qHTS) was used to det. IC50s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the max. affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compds. with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compd. interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quant. anal. of structure-activity relationships (>100K compds.) in addressing important questions such as a target's druggability.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvVWnt7o%253D&md5=aac37056be38a2c0ed06098d2e2f142a
  27. 27
    Fahey, R. C., Hunt, J. S., and Windham, G. C. (1977) On the cysteine and cystine content of proteins. Differences between intracellular and extracellular proteins J. Mol. Evol. 10, 155– 160
    Google Scholar
    There is no corresponding record for this reference.
  28. 28
    Goerke, A. R., Loening, A. M., Gambhir, S. S., and Swartz, J. R. (2008) Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase Metab. Eng. 10, 187– 200
    Google Scholar
    28
    Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
    Goerke, Aaron R.; Loening, Andreas M.; Gambhir, Sanjiv S.; Swartz, James R.
    Metabolic Engineering (2008), 10 (3-4), 187-200CODEN: MEENFM; ISSN:1096-7176. (Elsevier Inc.)
    Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc prodn. using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biol. provides the freedom to control both the catalyst and chem. compns. in biol. reactions, and we capitalized on this to produce large amts. of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell ext. source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metab., and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×1024 photons/s/mol, the highest reported activity for any characterized luciferase.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1ektbc%253D&md5=1f54e550163e57beeae2a131e48a513c
  29. 29
    Inouye, S. and Sasaki, S. (2007) Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris Protein Expression Purif. 56, 261– 268
    Google Scholar
    29
    Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris
    Inouye, Satoshi; Sasaki, Satoko
    Protein Expression and Purification (2007), 56 (2), 261-268CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)
    The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35 kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19KOLase, expressed as inclusion bodies, was solubilized with 6 M urea and purified by urea-nickel chelate affinity chromatog. The yield of 19kOLase was 16 mg from 400 mL of cultured cells. 19kOLase in 6 M urea could be refolded rapidly by diln. with 50 mM Tris-HCl (pH 7.8)-10 mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of org. solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1 μM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1anurrL&md5=0e4fd0d955d54530c24027053fb1cc18
  30. 30
    Wong, G. G., Witek-Giannotti, J., Hewick, R. M., Clark, S. C., and Ogawa, M. (1988) Interleukin 6: identification as a hematopoietic colony-stimulating factor Behring Inst. Mitt. 83, 40– 47
    Google Scholar
    30
    Interleukin 6: identification as a hematopoietic colony-stimulating factor
    Wong, G. G.; Witek-Giannotti, J.; Hewick, R. M.; Clark, S. C.; Ogawa, M.
    Behring Institute Mitteilungen (1988), 83 (), 40-7CODEN: BHIMA2; ISSN:0301-0457.
    A cDNA encoding a novel myeloid colony-stimulating activity from a human T cell line was identified and mol. cloned. This cytokine proved to be identical to the factor currently known as interleukin (IL)-6, thereby demonstrating effects of IL-6 with hematopoietic target cells. In addn. to its ability to support murine granulocyte-macrophage colony formation, IL-6 acted synergistically with IL-3 in both the murine and human systems in support of colony formation by the primitive blast cell colony forming cell. This multitude of biol. activities suggests that IL-6 plays a prominent role within a network of cytokines in integrating the different arms of the host response to infection.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXitFSmur8%253D&md5=b253f52b9e95b28e7ca725a9ef526756
  31. 31
    Lippincott-Schwartz, J., Roberts, T. H., and Hirschberg, K. (2000) Secretory protein trafficking and organelle dynamics in living cells Annu. Rev. Cell Dev. Biol. 16, 557– 589
    Google Scholar
    31
    Secretory protein trafficking and organelle dynamics in living cells
    Lippincott-Schwartz, Jennifer; Roberts, Theresa H.; Hirschberg, Koret
    Annual Review of Cell and Developmental Biology (2000), 16 (), 557-589CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)
    A review with many refs. Green fluorescent protein chimerae acting as reporters for protein localization and trafficking within the secretory membrane system of living cells have been used in a wide variety of applications, including time-lapse imaging, double-labeling, energy transfer, quantitation, and photobleaching expts. Results from this work are clarifying the steps involved in the formation, translocation, and fusion of transport intermediates; the organization and biogenesis of organelles; and the mechanisms of protein retention, sorting, and recycling in the secretory pathway. In so doing, they are broadening our thinking about the temporal and spatial relationships among secretory organelles and the membrane trafficking pathways that operate between them.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXpvFyq&md5=b5cf93a3d1fd4b750c843c43780bd567
  32. 32
    Simmons, S. O. (2011) Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing Comb. Chem. High Throughput Screening 14, 688– 702
    Google Scholar
    32
    Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing
    Simmons, Steven O.
    Combinatorial Chemistry & High Throughput Screening (2011), 14 (8), 688-702CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
    Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chems. currently in com. use. In addn., there are social and ethical pressures to reduce, refine and replace animal testing in toxicol. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chem. toxicants. Crit. to this vision, assays that measure toxicity pathways assocd. with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biol. activities in a quant. and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assays formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKqtrnI&md5=1224daf1cf9b892c342b3c87d13c478b
  33. 33
    Rehemtulla, A., Taneja, N., and Ross, B. D. (2004) Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event Mol. Imaging 3, 63– 68
    Google Scholar
    33
    Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event
    Rehemtulla, Alnawaz; Taneja, Neelam; Ross, Brian D.
    Molecular Imaging (2004), 3 (1), 63-68CODEN: MIOMBP; ISSN:1535-3508. (MIT Press)
    Inactivation of p53 is one of the most frequent mol. events in neoplastic transformation. Approx. 60% of all human tumors have mutations in both p53 alleles. Wild-type p53 activity is regulated in large part by the proteosome-dependent degrdn. of p53, resulting in a short p53 half-life in unstressed and untransformed cells. Activation of p53 by a variety of stimuli, including DNA damage induced by genotoxic drugs or radiation, is accomplished by stabilization of wild-type p53. The stabilized and active p53 can result in either cell-cycle arrest or apoptosis. Surprisingly, the majority of tumor-assocd., inactivating p53 mutations also result in p53 accumulation. Thus, constitutive elevation of p53 levels in cells is a reliable measure of p53 inactivation, whereas transiently increased p53 levels reflect a recent genotoxic stress. In order to facilitate noninvasive imaging of p53 accumulation, the authors here describe the construction of a p53-luciferase fusion protein. Induction of DNA damage in cells expressing, the fusion protein resulted in a time-dependent accumulation of the fusion that was noninvasively detected using bioluminescence imaging and validated by Western blot anal. The p53-Luc protein retains p53 function because its expression in HCT116 cells lacking functional p53 resulted in activation of p21 expression as well as induction of apoptosis in response to a DNA damaging event. Employed in a transgenic animal model, the proposed p53-reporter fusion protein will be useful for studying p53 activation in response to exposure to DNA-damaging carcinogenic agents. It could also be used to study p53 stabilization as a result of inactivating p53 mutations. Such studies will further the understanding of p53's role as the "guardian of the genome" and its function in tumorigenesis.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkslGjsLc%253D&md5=e3a74f1ffe9966213c680a7668f356e3
  34. 34
    Horn, H. F. and Vousden, K. H. (2007) Coping with stress: multiple ways to activate p53 Oncogene 26, 1306– 1316
    Google Scholar
    34
    Coping with stress: multiple ways to activate p53
    Horn, H. F.; Vousden, K. H.
    Oncogene (2007), 26 (9), 1306-1316CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
    A review. Over the years, p53 has been shown to sit at the center of an increasingly complex web of incoming stress signals and outgoing effector pathways. The no. and diversity of stress signals that lead to p53 activation illustrates the breadth of p53's remit - responding to a wide variety of potentially oncogenic insults to prevent tumor development. Interestingly, different stress signals can use different and independent pathways to activate p53, and there is some evidence that different stress signals can mediate different responses. How each of the responses to p53 contributes to inhibition of malignant progression is beginning to be clarified, with the hope that identification of responses that are key to tumor suppression will allow a more focused and effective search for new therapeutic targets. In this review, we will highlight some recently identified roles for p53 in tumor suppression, and discuss some of the numerous mechanisms through which p53 can be regulated and activated.
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXitVKhsLY%253D&md5=2d7b83d1eee3c060185f2441868ed16d
  35. 35
    Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion Nature 308, 693– 698
    Google Scholar
    35
    The role of protein kinase C in cell surface signal transduction and tumor promotion
    Nishizuka, Yasutomi
    Nature (London, United Kingdom) (1984), 308 (5961), 693-8CODEN: NATUAS; ISSN:0028-0836.
    A review with 80 refs. on the title role of protein kinase C [9026-43-1].
    >> More from SciFinder ®
    https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXitVCmtrk%253D&md5=33d139697753d3f7e10417573e131ff9
  36. 36
    Htun, H., Barsony, J., Renyi, I., Gould, D. L., and Hager, G. L. (1996) Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera Proc. Natl. Acad. Sci. U.S.A. 93, 4845– 4850
    Google Scholar
    There is no corresponding record for this reference.
  37. 37
    Nakajima, Y., Yamazaki, T., Nishii, S., Noguchi, T., Hoshino, H., Niwa, K., Viviani, V. R., and Ohmiya, Y. (2010) Enhanced beetle luciferase for high-resolution bioluminescence imaging PLoS One 5, e10011
    Google Scholar
    There is no corresponding record for this reference.
  38. 38
    Suzuki, T., Kondo, C., Kanamori, T., and Inouye, S. (2011) Video rate bioluminescence imaging of secretory proteins in living cells: localization, secretory frequency, and quantification Anal. Biochem. 415, 182– 189
    Google Scholar
    There is no corresponding record for this reference.

Cited By

Click to copy section linkSection link copied!

This article is cited by 1154 publications.

  1. Anneliese M. M. Gest, Ayse Z. Sahan, Yanghao Zhong, Wei Lin, Sohum Mehta, Jin Zhang. Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals. Chemical Reviews 2024, 124 (22) , 12573-12660. https://doi.org/10.1021/acs.chemrev.4c00293
  2. Giulia Tedeschi, Mariana X. Navarro, Lorenzo Scipioni, Tanvi K. Sondhi, Jennifer A. Prescher, Michelle A. Digman. Monitoring Macrophage Polarization with Gene Expression Reporters and Bioluminescence Phasor Analysis. Chemical & Biomedical Imaging 2024, 2 (11) , 765-774. https://doi.org/10.1021/cbmi.4c00049
  3. Yinghui Yang, Akihito Inoue, Takanobu Yasuda, Hiroshi Ueda, Bo Zhu, Tetsuya Kitaguchi. BRET Nano Q-Body: A Nanobody-Based Ratiometric Bioluminescent Immunosensor for Point-of-Care Testing. ACS Sensors 2024, 9 (11) , 5955-5965. https://doi.org/10.1021/acssensors.4c01800
  4. Paramesh K. Ramaraj, Mugdha Pol, Samuel L. Scinto, Xinqiao Jia, Joseph M. Fox. Covalent Attachment of Functional Proteins to Microfiber Surfaces via a General Strategy for Site-Selective Tetrazine Ligation. ACS Applied Materials & Interfaces 2024, 16 (46) , 63195-63206. https://doi.org/10.1021/acsami.4c12609
  5. Yoo-Hong Min, Yoonseo Hong, Cheol-Hee Kim, Kyung-Ho Lee, Yong-Beom Shin, Ju-Young Byun. Split Probe-Induced Protein Translational Amplification for Nucleic Acid Detection. ACS Applied Bio Materials 2024, Article ASAP.
  6. Antoine Lévrier, Julien Capin, Pauline Mayonove, Ioannis-Ilie Karpathakis, Peter Voyvodic, Angelique DeVisch, Ana Zuniga, Martin Cohen-Gonsaud, Stéphanie Cabantous, Vincent Noireaux, Jerome Bonnet. Split Reporters Facilitate Monitoring of Gene Expression and Peptide Production in Linear Cell-Free Transcription–Translation Systems. ACS Synthetic Biology 2024, 13 (10) , 3119-3127. https://doi.org/10.1021/acssynbio.4c00353
  7. Yanian Xiong, Yingtung Lo, Huizhu Song, Jianzhong Lu. Development of a Self-Luminescent Living Bioreactor for Enhancing Photodynamic Therapy in Breast Cancer. Bioconjugate Chemistry 2024, 35 (8) , 1269-1282. https://doi.org/10.1021/acs.bioconjchem.4c00334
  8. Lingfei Wang, Hanfeng Lin, Bin Yang, Xiqian Jiang, Jianwei Chen, Sandipan Roy Chowdhury, Ninghui Cheng, Paul A. Nakata, David M. Lonard, Meng. C. Wang, Jin Wang. Development of a Novel Amplifiable System to Quantify Hydrogen Peroxide in Living Cells. Journal of the American Chemical Society 2024, 146 (32) , 22396-22404. https://doi.org/10.1021/jacs.4c05366
  9. Hanfeng Lin, Kristin Riching, May Poh Lai, Dong Lu, Ran Cheng, Xiaoli Qi, Jin Wang. Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools for Monitoring Targeted Protein Degradation. ACS Medicinal Chemistry Letters 2024, 15 (8) , 1367-1375. https://doi.org/10.1021/acsmedchemlett.4c00271
  10. Brian L. Zhong, Jeandele M. Elliot, Pengli Wang, Hongquan Li, R. Nelson Hall, Bo Wang, Manu Prakash, Alexander R. Dunn. Split Luciferase Molecular Tension Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems. ACS Sensors 2024, 9 (7) , 3489-3495. https://doi.org/10.1021/acssensors.3c02664
  11. Eleonora Comeo, Joëlle Goulding, Chia-Yang Lin, Marleen Groenen, Jeanette Woolard, Nicholas D. Kindon, Clare R. Harwood, Simon Platt, Stephen J. Briddon, Laura E. Kilpatrick, Peter J. Scammells, Stephen J. Hill, Barrie Kellam. Ligand-Directed Labeling of the Adenosine A1 Receptor in Living Cells. Journal of Medicinal Chemistry 2024, 67 (14) , 12099-12117. https://doi.org/10.1021/acs.jmedchem.4c00835
  12. Atsushi Ogawa, Masahiro Fujikawa, Kazuki Onishi, Hajime Takahashi. Cell-Free Biosensors Based on Modular Eukaryotic Riboswitches That Function in One Pot at Ambient Temperature. ACS Synthetic Biology 2024, 13 (7) , 2238-2245. https://doi.org/10.1021/acssynbio.4c00341
  13. Lara Toy, Max E. Huber, Minhee Lee, Ana Alonso Bartolomé, Natalia V. Ortiz Zacarías, Sherif Nasser, Stephan Scholl, Darius P. Zlotos, Yasmine M. Mandour, Laura H. Heitman, Martyna Szpakowska, Andy Chevigné, Matthias Schiedel. Fluorophore-Labeled Pyrrolones Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR1. ACS Pharmacology & Translational Science 2024, 7 (7) , 2080-2092. https://doi.org/10.1021/acsptsci.4c00182
  14. Juan Sanz García, Rosa Maskri, Alexander Mitrushchenkov, Loïc Joubert-Doriol. Optimizing Conical Intersections without Explicit Use of Non-Adiabatic Couplings. Journal of Chemical Theory and Computation 2024, 20 (13) , 5643-5654. https://doi.org/10.1021/acs.jctc.4c00326
  15. Angelica Rose Galvan, Christopher M. Green, Shelby L. Hooe, Esra Oktay, Meghna Thakur, Sebastián A. Díaz, Remi Veneziano, Igor L. Medintz, Divita Mathur. Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System. ACS Applied Nano Materials 2024, 7 (11) , 12891-12902. https://doi.org/10.1021/acsanm.4c01456
  16. Georg Rueedi, Philippe Panchaud, Astrid Friedli, Jean-Luc Specklin, Christian Hubschwerlen, Anne-Catherine Blumstein, Patrick Caspers, Michel Enderlin-Paput, Loïc Jacob, Christopher Kohl, Hans H. Locher, Philippe Pfaff, Christine Schmitt, Peter Seiler, Daniel Ritz. Discovery and Structure–Activity Relationship of Cadazolid: A First-In-Class Quinoxolidinone Antibiotic for the Treatment of Clostridioides difficile Infection. Journal of Medicinal Chemistry 2024, 67 (11) , 9465-9484. https://doi.org/10.1021/acs.jmedchem.4c00558
  17. Francesco Russo, Beatrice Civili, Nicolas Winssinger. Bright Red Bioluminescence from Semisynthetic NanoLuc (sNLuc). ACS Chemical Biology 2024, 19 (5) , 1035-1039. https://doi.org/10.1021/acschembio.4c00033
  18. Shengyu Zhao, Ying Xiong, Ranganayakulu Sunnapu, Yiyu Zhang, Xiaodong Tian, Hui-wang Ai. Bioluminescence Imaging of Potassium Ion Using a Sensory Luciferin and an Engineered Luciferase. Journal of the American Chemical Society 2024, 146 (19) , 13406-13416. https://doi.org/10.1021/jacs.4c02473
  19. Hannah Vogt, Patrick Shinkwin, Max E. Huber, Nico Staffen, Harald Hübner, Peter Gmeiner, Matthias Schiedel, Dorothee Weikert. Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1. ACS Pharmacology & Translational Science 2024, 7 (5) , 1533-1545. https://doi.org/10.1021/acsptsci.4c00086
  20. Taylor B. Dolberg, Taylor F. Gunnels, Te Ling, Kelly A. Sarnese, John D. Crispino, Joshua N. Leonard. Building Synthetic Biosensors Using Red Blood Cell Proteins. ACS Synthetic Biology 2024, 13 (4) , 1273-1289. https://doi.org/10.1021/acssynbio.3c00754
  21. Jakob Gleixner, Sergei Kopanchuk, Lukas Grätz, Maris-Johanna Tahk, Tõnis Laasfeld, Santa Veikšina, Carina Höring, Albert O. Gattor, Laura J. Humphrys, Christoph Müller, Nataliya Archipowa, Johannes Köckenberger, Markus R. Heinrich, Roger Jan Kutta, Ago Rinken, Max Keller. Illuminating Neuropeptide Y Y4 Receptor Binding: Fluorescent Cyclic Peptides with Subnanomolar Binding Affinity as Novel Molecular Tools. ACS Pharmacology & Translational Science 2024, 7 (4) , 1142-1168. https://doi.org/10.1021/acsptsci.4c00013
  22. Eva A. van Aalen, Joep J. J. Lurvink, Leandra Vermeulen, Benice van Gerven, Yan Ni, Remco Arts, Maarten Merkx. Turning Antibodies into Ratiometric Bioluminescent Sensors for Competition-Based Homogeneous Immunoassays. ACS Sensors 2024, 9 (3) , 1401-1409. https://doi.org/10.1021/acssensors.3c02478
  23. Ethan M. Jones, Yang Su, Chris Sander, Quincey A. Justman, Michael Springer, Pamela A. Silver. LanTERN: A Fluorescent Sensor That Specifically Responds to Lanthanides. ACS Synthetic Biology 2024, 13 (3) , 958-962. https://doi.org/10.1021/acssynbio.3c00600
  24. Mark A. Klein, Sergey Lazarev, Charles Gervasi, Cristopher Cowan, Thomas Machleidt, Rachel Friedman Ohana. Luciferase Calibrants Enable Absolute Quantitation of Bioluminescence Power. ACS Measurement Science Au 2023, 3 (6) , 496-503. https://doi.org/10.1021/acsmeasuresciau.3c00036
  25. Yi-Sheng Lu, Ruhan Fan, Willem Vugs, Lieuwe Biewenga, Ziming Zhou, Sanahan Vijayakumar, Maarten Merkx, Michael J. Sailor. Harnessing the Materials Chemistry of Mesoporous Silicon Nanoparticles to Prepare “Armor-Clad” Enzymes. Chemistry of Materials 2023, 35 (23) , 10247-10257. https://doi.org/10.1021/acs.chemmater.3c02637
  26. Masatoshi Eguchi, Hideaki Yoshimura, Yoshibumi Ueda, Takeaki Ozawa. Split Luciferase-Fragment Reconstitution for Unveiling RNA Localization and Dynamics in Live Cells. ACS Sensors 2023, 8 (11) , 4055-4063. https://doi.org/10.1021/acssensors.3c01080
  27. Philipp Kemp, Wadim Weber, Charlotte Desczyk, Marwan Kaufmann, Josefine Panthel, Theresa Wörmann, Viktor Stein. Dissecting the Permeability of the Escherichia coli Cell Envelope to a Small Molecule Using Tailored Intensiometric Fluorescent Protein Sensors. ACS Omega 2023, 8 (42) , 39562-39569. https://doi.org/10.1021/acsomega.3c05405
  28. Dawson B. Ling, William Nguyen, Oliver Looker, Zahra Razook, Kirsty McCann, Alyssa E. Barry, Christian Scheurer, Sergio Wittlin, Mufuliat Toyin Famodimu, Michael J Delves, Hayley E. Bullen, Brendan S. Crabb, Brad E. Sleebs, Paul R. Gilson. A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite’s PI4KIIIB Enzyme. ACS Infectious Diseases 2023, 9 (9) , 1695-1710. https://doi.org/10.1021/acsinfecdis.3c00138
  29. Whitney K. Lieberman, Zachary A. Brown, Daniel S. Kantner, Yihang Jing, Emily Megill, Nya D. Evans, McKenna C. Crawford, Isita Jhulki, Carissa Grose, Jane E. Jones, Nathaniel W. Snyder, Jordan L. Meier. Chemoproteomics Yields a Selective Molecular Host for Acetyl-CoA. Journal of the American Chemical Society 2023, 145 (30) , 16899-16905. https://doi.org/10.1021/jacs.3c05489
  30. Max E. Huber, Silas Wurnig, Lara Toy, Corinna Weiler, Nicole Merten, Evi Kostenis, Finn K. Hansen, Matthias Schiedel. Fluorescent Ligands Enable Target Engagement Studies for the Intracellular Allosteric Binding Site of the Chemokine Receptor CXCR2. Journal of Medicinal Chemistry 2023, 66 (14) , 9916-9933. https://doi.org/10.1021/acs.jmedchem.3c00769
  31. Vasilisa V. Krasitskaya, Maxim K. Efremov, Ludmila A. Frank. Luciferase NLuc Site-Specific Conjugation to Generate Reporters for In Vitro Assays. Bioconjugate Chemistry 2023, 34 (7) , 1282-1289. https://doi.org/10.1021/acs.bioconjchem.3c00165
  32. Ruslan Gibadullin, Brian P. Cary, Samuel H. Gellman. Differential Responses of the GLP-1 and GLP-2 Receptors to N-Terminal Modification of a Dual Agonist. Journal of the American Chemical Society 2023, 145 (22) , 12105-12114. https://doi.org/10.1021/jacs.3c01628
  33. Junbin Li, Na Wang, Mengyi Xiong, Min Dai, Cheng Xie, Qianqian Wang, Ke Quan, Yibo Zhou, Zhihe Qing. A Reaction-Based Ratiometric Bioluminescent Platform for Point-of-Care and Quantitative Detection Using a Smartphone. Analytical Chemistry 2023, 95 (18) , 7142-7149. https://doi.org/10.1021/acs.analchem.2c05422
  34. Anhuy Pham, Shane Bassett, Wilfred Chen, Nancy A. Da Silva. Assembly of Metabolons in Yeast Using Cas6-Mediated RNA Scaffolding. ACS Synthetic Biology 2023, 12 (4) , 1164-1174. https://doi.org/10.1021/acssynbio.2c00650
  35. Lai Wei, Kaijing Xiang, Hongjian Kang, Yancheng Yu, Hongjie Fan, Han Zhou, Tao Hou, Yonglin Ge, Jixia Wang, Zhimou Guo, Yang Chen, Yaopeng Zhao, Xinmiao Liang. Development and Characterization of Fluorescent Probes for the G Protein-Coupled Receptor 35. ACS Medicinal Chemistry Letters 2023, 14 (4) , 411-416. https://doi.org/10.1021/acsmedchemlett.2c00461
  36. Jie Yu, Yan Zhang, Yanping Zhao, Xuxiang Zhang, Hongqiang Ren. Highly Sensitive and Selective Detection of Inorganic Phosphates in the Water Environment by Biosensors Based on Bioluminescence Resonance Energy Transfer. Analytical Chemistry 2023, 95 (11) , 4904-4913. https://doi.org/10.1021/acs.analchem.2c04748
  37. Peter Braun, Rene Raab, Joachim J. Bugert, Simone Braun. Recombinant Reporter Phage rTUN1::nLuc Enables Rapid Detection and Real-Time Antibiotic Susceptibility Testing of Klebsiella pneumoniae K64 Strains. ACS Sensors 2023, 8 (2) , 630-639. https://doi.org/10.1021/acssensors.2c01822
  38. Anna C. Love, Donald R. Caldwell, Bethany Kolbaba-Kartchner, Katherine M. Townsend, Lila P. Halbers, Zi Yao, Caroline K. Brennan, Joseph Ivanic, Tanya Hadjian, Jeremy H. Mills, Martin J. Schnermann, Jennifer A. Prescher. Red-Shifted Coumarin Luciferins for Improved Bioluminescence Imaging. Journal of the American Chemical Society 2023, 145 (6) , 3335-3345. https://doi.org/10.1021/jacs.2c07220
  39. Lan Mi, Qikun Yu, Aruni P. K. K. Karunanayake Mudiyanselage, Rigumula Wu, Zhining Sun, Ru Zheng, Kewei Ren, Mingxu You. Genetically Encoded RNA-Based Bioluminescence Resonance Energy Transfer (BRET) Sensors. ACS Sensors 2023, 8 (1) , 308-316. https://doi.org/10.1021/acssensors.2c02213
  40. Atsushi Ogawa, Honami Inoue, Yu Itoh, Hajime Takahashi. Facile Expansion of the Variety of Orthogonal Ligand/Aptamer Pairs for Artificial Riboswitches. ACS Synthetic Biology 2023, 12 (1) , 35-42. https://doi.org/10.1021/acssynbio.2c00475
  41. Jingxin Liu, Chenchen Ge, Ling Zha, Ligen Lin, Rongsong Li. Simple Nano-Luciferase-Based Assay for the Rapid and High-Throughput Detection of SARS-CoV-2 3C-Like Protease. Analytical Chemistry 2023, 95 (2) , 714-719. https://doi.org/10.1021/acs.analchem.2c02590
  42. Dimitra Apostolidou, Pan Zhang, Weitao Yang, Piotr E. Marszalek. Mechanical Unfolding and Refolding of NanoLuc via Single-Molecule Force Spectroscopy and Computer Simulations. Biomacromolecules 2022, 23 (12) , 5164-5178. https://doi.org/10.1021/acs.biomac.2c00997
  43. Shikha S. Chauhan, Nick J. Marotta, Anna C. Karls, Emily E. Weinert. Binding of 2′,3′-Cyclic Nucleotide Monophosphates to Bacterial Ribosomes Inhibits Translation. ACS Central Science 2022, 8 (11) , 1518-1526. https://doi.org/10.1021/acscentsci.2c00681
  44. Kathryn A. Hufziger, Emma L. Farquharson, Brenda G. Werner, Qingmin Chen, Julie M. Goddard, Sam R. Nugen. In Vivo Capsid Engineering of Bacteriophages for Oriented Surface Conjugation. ACS Applied Bio Materials 2022, 5 (11) , 5104-5112. https://doi.org/10.1021/acsabm.2c00428
  45. Lukas Grätz, Christoph Müller, Andrea Pegoli, Lisa Schindler, Günther Bernhardt, Timo Littmann. Insertion of Nanoluc into the Extracellular Loops as a Complementary Method To Establish BRET-Based Binding Assays for GPCRs. ACS Pharmacology & Translational Science 2022, 5 (11) , 1142-1155. https://doi.org/10.1021/acsptsci.2c00162
  46. Mohammad Javad Afshari, Cang Li, Jianfeng Zeng, Jiabin Cui, Shuwang Wu, Mingyuan Gao. Self-illuminating NIR-II bioluminescence imaging probe based on silver sulfide quantum dots. ACS Nano 2022, 16 (10) , 16824-16832. https://doi.org/10.1021/acsnano.2c06667
  47. Maylis Boitet, Hyeju Eun, Asma Achek, Virgínia Carla de Almeida Falcão, Vincent Delorme, Regis Grailhe. Biolum’ RGB: A Low-Cost, Versatile, and Sensitive Bioluminescence Imaging Instrument for a Broad Range of Users. ACS Sensors 2022, 7 (9) , 2556-2566. https://doi.org/10.1021/acssensors.2c00457
  48. Tino W. Sanchez, Michael H. Ronzetti, Ashley E. Owens, Maria Antony, Ty Voss, Eric Wallgren, Daniel Talley, Krishna Balakrishnan, Sebastian E. Leyes Porello, Ganesha Rai, Juan J. Marugan, Samuel G. Michael, Bolormaa Baljinnyam, Noel Southall, Anton Simeonov, Mark J. Henderson. Real-Time Cellular Thermal Shift Assay to Monitor Target Engagement. ACS Chemical Biology 2022, 17 (9) , 2471-2482. https://doi.org/10.1021/acschembio.2c00334
  49. Anil Mathew Tharappel, Zhong Li, Yan Chun Zhu, Xiangmeng Wu, Sudha Chaturvedi, Qing-Yu Zhang, Hongmin Li. Calcimycin Inhibits Cryptococcus neoformans In Vitro and In Vivo by Targeting the Prp8 Intein Splicing. ACS Infectious Diseases 2022, 8 (9) , 1851-1868. https://doi.org/10.1021/acsinfecdis.2c00137
  50. Lara Toy, Max E. Huber, Maximilian F. Schmidt, Dorothee Weikert, Matthias Schiedel. Fluorescent Ligands Targeting the Intracellular Allosteric Binding Site of the Chemokine Receptor CCR2. ACS Chemical Biology 2022, 17 (8) , 2142-2152. https://doi.org/10.1021/acschembio.2c00263
  51. Virginia A. Kincaid, Hui Wang, Casey A. Sondgeroth, Emily A. Torio, Valerie T. Ressler, Connor Fitzgerald, Mary P. Hall, Robin Hurst, Monika G. Wood, Julia K. Gilden, Thomas A. Kirkland, Dan Lazar, Hsu Chia-Chang, Lance P. Encell, Thomas Machleidt, Wenhui Zhou, Melanie L. Dart. Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides. ACS Chemical Biology 2022, 17 (8) , 2179-2187. https://doi.org/10.1021/acschembio.2c00306
  52. Rebeka C. Fanti, Stanley N. S. Vasconcelos, Carolina M. C. Catta-Preta, Jaryd R. Sullivan, Gustavo P. Riboldi, Caio V. dos Reis, Priscila Z. Ramos, Aled M. Edwards, Marcel A. Behr, Rafael M. Couñago. A Target Engagement Assay to Assess Uptake, Potency, and Retention of Antibiotics in Living Bacteria. ACS Infectious Diseases 2022, 8 (8) , 1449-1467. https://doi.org/10.1021/acsinfecdis.2c00073
  53. Ying Xiong, Yiyu Zhang, Zefan Li, Md Shamim Reza, Xinyu Li, Xiaodong Tian, Hui-wang Ai. Engineered Amber-Emitting Nano Luciferase and Its Use for Immunobioluminescence Imaging In Vivo. Journal of the American Chemical Society 2022, 144 (31) , 14101-14111. https://doi.org/10.1021/jacs.2c02320
  54. Dan Sindhikara Jennifer Johnston . Roles of Conformations on Predictions of Peptide Properties. , 103-135. https://doi.org/10.1021/bk-2022-1417.ch004
  55. Alexander Gräwe, Maarten Merkx, Viktor Stein. iFLinkC-X: A Scalable Framework to Assemble Bespoke Genetically Encoded Co-polymeric Linkers of Variable Lengths and Amino Acid Composition. Bioconjugate Chemistry 2022, 33 (7) , 1415-1421. https://doi.org/10.1021/acs.bioconjchem.2c00250
  56. Nikki McArthur, Carlos Cruz-Teran, Apoorva Thatavarty, Gregory T. Reeves, Balaji M. Rao. Experimental and Analytical Framework for “Mix-and-Read” Assays Based on Split Luciferase. ACS Omega 2022, 7 (28) , 24551-24560. https://doi.org/10.1021/acsomega.2c02319
  57. Xuan Yang, Rebekah J. Dickmander, Armin Bayati, Sharon A. Taft-Benz, Jeffery L. Smith, Carrow I. Wells, Emily A. Madden, Jason W. Brown, Erik M. Lenarcic, Boyd L. Yount, Jr, Edcon Chang, Alison D. Axtman, Ralph S. Baric, Mark T. Heise, Peter S. McPherson, Nathaniel J. Moorman, Timothy M. Willson. Host Kinase CSNK2 is a Target for Inhibition of Pathogenic SARS-like β-Coronaviruses. ACS Chemical Biology 2022, 17 (7) , 1937-1950. https://doi.org/10.1021/acschembio.2c00378
  58. Masaharu Somiya, Shun’ichi Kuroda. Engineering of Extracellular Vesicles for Small Molecule-Regulated Cargo Loading and Cytoplasmic Delivery of Bioactive Proteins. Molecular Pharmaceutics 2022, 19 (7) , 2495-2505. https://doi.org/10.1021/acs.molpharmaceut.2c00192
  59. Claire M. S. Michielsen, Eva A. van Aalen, Maarten Merkx. Ratiometric Bioluminescent Zinc Sensor Proteins to Quantify Serum and Intracellular Free Zn2+. ACS Chemical Biology 2022, 17 (6) , 1567-1576. https://doi.org/10.1021/acschembio.2c00227
  60. Hope Adamson, Modupe O. Ajayi, Kate E. Gilroy, Michael J. McPherson, Darren C. Tomlinson, Lars J. C. Jeuken. Rapid Quantification of C. difficile Glutamate Dehydrogenase and Toxin B (TcdB) with a NanoBiT Split-Luciferase Assay. Analytical Chemistry 2022, 94 (23) , 8156-8163. https://doi.org/10.1021/acs.analchem.1c05206
  61. Yuna Nakagawa, Jan Vincent V. Arafiles, Yoshimasa Kawaguchi, Ikuhiko Nakase, Hisaaki Hirose, Shiroh Futaki. Stearylated Macropinocytosis-Inducing Peptides Facilitating the Cellular Uptake of Small Extracellular Vesicles. Bioconjugate Chemistry 2022, 33 (5) , 869-880. https://doi.org/10.1021/acs.bioconjchem.2c00113
  62. Wenhua Li, Zhao Ma, Jiwei Chen, Gaopan Dong, Lupei Du, Minyong Li. Discovery of Environment-Sensitive Fluorescent Ligands of β-Adrenergic Receptors for Cell Imaging and NanoBRET Assay. Analytical Chemistry 2022, 94 (19) , 7021-7028. https://doi.org/10.1021/acs.analchem.1c05646
  63. Yuki Ohmuro-Matsuyama, Tadaomi Furuta, Hayato Matsui, Masaki Kanai, Hiroshi Ueda. Miniaturization of Bright Light-Emitting Luciferase ALuc: picALuc. ACS Chemical Biology 2022, 17 (4) , 864-872. https://doi.org/10.1021/acschembio.1c00897
  64. Elbegduuren Erdenee, Alice Y. Ting. A Dual-Purpose Real-Time Indicator and Transcriptional Integrator for Calcium Detection in Living Cells. ACS Synthetic Biology 2022, 11 (3) , 1086-1095. https://doi.org/10.1021/acssynbio.1c00597
  65. Mariko Orioka, Masatoshi Eguchi, Yuki Mizui, Yuma Ikeda, Akihiro Sakama, Qiaojing Li, Hideaki Yoshimura, Takeaki Ozawa, Daniel Citterio, Yuki Hiruta. A Series of Furimazine Derivatives for Sustained Live-Cell Bioluminescence Imaging and Application to the Monitoring of Myogenesis at the Single-Cell Level. Bioconjugate Chemistry 2022, 33 (3) , 496-504. https://doi.org/10.1021/acs.bioconjchem.2c00035
  66. Linggang Zheng, Yang Tan, Yucan Hu, Juntao Shen, Zepeng Qu, Xianbo Chen, Chun Loong Ho, Elaine Lai-Han Leung, Wei Zhao, Lei Dai. CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species. ACS Synthetic Biology 2022, 11 (1) , 464-472. https://doi.org/10.1021/acssynbio.1c00543
  67. Ssu-Tzu Tsai, Wen-Jui Cheng, Qian-Xian Zhang, Yi-Chun Yeh. Gold-Specific Biosensor for Monitoring Wastewater Using Genetically Engineered Cupriavidus metallidurans CH34. ACS Synthetic Biology 2021, 10 (12) , 3576-3582. https://doi.org/10.1021/acssynbio.1c00520
  68. Mihris Ibnu Saleem Naduthodi, Christian Südfeld, Emmanouil Klimis Avitzigiannis, Nicola Trevisan, Eduard van Lith, Javier Alcaide Sancho, Sarah D’Adamo, Maria Barbosa, John van der Oost. Comprehensive Genome Engineering Toolbox for Microalgae Nannochloropsis oceanica Based on CRISPR-Cas Systems. ACS Synthetic Biology 2021, 10 (12) , 3369-3378. https://doi.org/10.1021/acssynbio.1c00329
  69. Shirley Liu, Yichi Su, Michael Z. Lin, John A. Ronald. Brightening up Biology: Advances in Luciferase Systems for in Vivo Imaging. ACS Chemical Biology 2021, 16 (12) , 2707-2718. https://doi.org/10.1021/acschembio.1c00549
  70. Taha Azad, Helena J. Janse van Rensburg, Jessica Morgan, Reza Rezaei, Mathieu J. F. Crupi, Rui Chen, Mina Ghahremani, Monire Jamalkhah, Nicole Forbes, Carolina Ilkow, John C. Bell. Luciferase-Based Biosensors in the Era of the COVID-19 Pandemic. ACS Nanoscience Au 2021, 1 (1) , 15-37. https://doi.org/10.1021/acsnanoscienceau.1c00009
  71. Mengying Deng, Jing Yuan, Haibin Yang, Xuli Wu, Xiaoyuan Wei, Yang Du, Garry Wong, Yuyong Tao, Gang Liu, Zongwen Jin, Jun Chu. A Genetically Encoded Bioluminescent System for Fast and Highly Sensitive Detection of Antibodies with a Bright Green Fluorescent Protein. ACS Nano 2021, 15 (11) , 17602-17612. https://doi.org/10.1021/acsnano.1c05164
  72. Marcel V. Alavi. OMA1 High-Throughput Screen Reveals Protease Activation by Kinase Inhibitors. ACS Chemical Biology 2021, 16 (11) , 2202-2211. https://doi.org/10.1021/acschembio.1c00350
  73. Muyang Guan, Mikael V. Garabedian, Marcel Leutenegger, Benjamin S. Schuster, Matthew C. Good, Daniel A. Hammer. Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates. Biochemistry 2021, 60 (42) , 3137-3151. https://doi.org/10.1021/acs.biochem.1c00373
  74. Mitsuru Hattori, Nae Sugiura, Tetsuichi Wazawa, Tomoki Matsuda, Takeharu Nagai. Ratiometric Bioluminescent Indicator for a Simple and Rapid Measurement of Thrombin Activity Using a Smartphone. Analytical Chemistry 2021, 93 (40) , 13520-13526. https://doi.org/10.1021/acs.analchem.1c02396
  75. Yu Pang, Hao Zhang, Hui-wang Ai. Genetically Encoded Fluorescent Redox Indicators for Unveiling Redox Signaling and Oxidative Toxicity. Chemical Research in Toxicology 2021, 34 (8) , 1826-1845. https://doi.org/10.1021/acs.chemrestox.1c00149
  76. Jonathan D. Mortison, Ivan Cornella-Taracido, Gireedhar Venkatchalam, Anthony W. Partridge, Nirodhini Siriwardana, Simon M. Bushell. Rapid Evaluation of Small Molecule Cellular Target Engagement with a Luminescent Thermal Shift Assay. ACS Medicinal Chemistry Letters 2021, 12 (8) , 1288-1294. https://doi.org/10.1021/acsmedchemlett.1c00276
  77. Álvaro Luque-Uría, Riikka Peltomaa, Tarja K. Nevanen, Henri O. Arola, Kristiina Iljin, Elena Benito-Peña, María C. Moreno-Bondi. Recombinant Peptide Mimetic NanoLuc Tracer for Sensitive Immunodetection of Mycophenolic Acid. Analytical Chemistry 2021, 93 (29) , 10358-10364. https://doi.org/10.1021/acs.analchem.1c02109
  78. Sim Yee Chen, Melissa Harrison, Eng Khoon Ng, Dominic Sauvageau, Anastasia Elias. Immobilized Reporter Phage on Electrospun Polymer Fibers for Improved Capture and Detection of Escherichia coli O157:H7. ACS Food Science & Technology 2021, 1 (6) , 1085-1094. https://doi.org/10.1021/acsfoodscitech.1c00101
  79. Hyukjun Choi, Soomin Eom, Han-ul Kim, Yoonji Bae, Hyun Suk Jung, Sebyung Kang. Load and Display: Engineering Encapsulin as a Modular Nanoplatform for Protein-Cargo Encapsulation and Protein-Ligand Decoration Using Split Intein and SpyTag/SpyCatcher. Biomacromolecules 2021, 22 (7) , 3028-3039. https://doi.org/10.1021/acs.biomac.1c00481
  80. Riho Takahashi, Takanobu Yasuda, Yuki Ohmuro-Matsuyama, Hiroshi Ueda. BRET Q-Body: A Ratiometric Quench-based Bioluminescent Immunosensor Made of Luciferase–Dye–Antibody Fusion with Enhanced Response. Analytical Chemistry 2021, 93 (21) , 7571-7578. https://doi.org/10.1021/acs.analchem.0c05217
  81. Brittany R. Benlian, Pavel E. Z. Klier, Kayli N. Martinez, Marie K. Schwinn, Thomas A. Kirkland, Evan W. Miller. Small Molecule–Protein Hybrid for Voltage Imaging via Quenching of Bioluminescence. ACS Sensors 2021, 6 (5) , 1857-1863. https://doi.org/10.1021/acssensors.1c00058
  82. Eleonora Comeo, Phuc Trinh, Anh T. Nguyen, Cameron J. Nowell, Nicholas D. Kindon, Mark Soave, Leigh A. Stoddart, Jonathan M. White, Stephen J. Hill, Barrie Kellam, Michelle L. Halls, Lauren T. May, Peter J. Scammells. Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A1 Receptor in Living Cells. Journal of Medicinal Chemistry 2021, 64 (10) , 6670-6695. https://doi.org/10.1021/acs.jmedchem.0c02067
  83. Kwangho Kim, Plamen P. Christov, Ian Romaine, Jianhua Tian, Somnath Jana, Alexander P. Lamers, Brendan F. Dutter, Toya Scaggs, Kyouk Jeon, Benjamin Guttentag, C. David Weaver, Craig W. Lindsley, Alex G. Waterson, Gary A. Sulikowski. Ten-Year Retrospective of the Vanderbilt Institute of Chemical Biology Chemical Synthesis Core. ACS Chemical Biology 2021, 16 (5) , 787-793. https://doi.org/10.1021/acschembio.0c00818
  84. Masaharu Somiya, Shun’ichi Kuroda. Real-Time Luminescence Assay for Cytoplasmic Cargo Delivery of Extracellular Vesicles. Analytical Chemistry 2021, 93 (13) , 5612-5620. https://doi.org/10.1021/acs.analchem.1c00339
  85. Mary P. Hall, Virginia A. Kincaid, Emily A. Jost, Thomas P. Smith, Robin Hurst, Stuart K. Forsyth, Connor Fitzgerald, Valerie T. Ressler, Kris Zimmermann, Dan Lazar, Monika G. Wood, Keith V. Wood, Thomas A. Kirkland, Lance P. Encell, Thomas Machleidt, Melanie L. Dart. Toward a Point-of-Need Bioluminescence-Based Immunoassay Utilizing a Complete Shelf-Stable Reagent. Analytical Chemistry 2021, 93 (12) , 5177-5184. https://doi.org/10.1021/acs.analchem.0c05074
  86. Yukino Itoh, Mitsuru Hattori, Tetsuichi Wazawa, Yoshiyuki Arai, Takeharu Nagai. Ratiometric Bioluminescent Indicator for Simple and Rapid Diagnosis of Bilirubin. ACS Sensors 2021, 6 (3) , 889-895. https://doi.org/10.1021/acssensors.0c02000
  87. Kaitlyn Bacon, Abigail Blain, John Bowen, Matthew Burroughs, Nikki McArthur, Stefano Menegatti, Balaji M. Rao. Quantitative Yeast–Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein–Protein Interactions. ACS Synthetic Biology 2021, 10 (3) , 505-514. https://doi.org/10.1021/acssynbio.0c00472
  88. Elisa Lázaro-Ibáñez, Farid N. Faruqu, Amer F. Saleh, Andreia M. Silva, Julie Tzu-Wen Wang, Janusz Rak, Khuloud T. Al-Jamal, Niek Dekker. Selection of Fluorescent, Bioluminescent, and Radioactive Tracers to Accurately Reflect Extracellular Vesicle Biodistribution in Vivo. ACS Nano 2021, 15 (2) , 3212-3227. https://doi.org/10.1021/acsnano.0c09873
  89. Aida Shahraki, Ali Işbilir, Berna Dogan, Martin J. Lohse, Serdar Durdagi, Necla Birgul-Iyison. Structural and Functional Characterization of Allatostatin Receptor Type-C of Thaumetopoea pityocampa, a Potential Target for Next-Generation Pest Control Agents. Journal of Chemical Information and Modeling 2021, 61 (2) , 715-728. https://doi.org/10.1021/acs.jcim.0c00985
  90. Andrea Peier, Lan Ge, Nicolas Boyer, John Frost, Ruchia Duggal, Kaustav Biswas, Scott Edmondson, Jeffrey D. Hermes, Lin Yan, Chad Zimprich, Ahmad Sadruddin, Hung Yi Kristal Kaan, Arun Chandramohan, Christopher J. Brown, Dawn Thean, Xue Er Lee, Tsz Ying Yuen, Fernando J. Ferrer-Gago, Charles W. Johannes, David P. Lane, Brad Sherborne, Cesear Corona, Matthew B. Robers, Tomi K. Sawyer, Anthony W. Partridge. NanoClick: A High Throughput, Target-Agnostic Peptide Cell Permeability Assay. ACS Chemical Biology 2021, 16 (2) , 293-309. https://doi.org/10.1021/acschembio.0c00804
  91. Adam D. Cotton, Duy P. Nguyen, Josef A. Gramespacher, Ian B. Seiple, James A. Wells. Development of Antibody-Based PROTACs for the Degradation of the Cell-Surface Immune Checkpoint Protein PD-L1. Journal of the American Chemical Society 2021, 143 (2) , 593-598. https://doi.org/10.1021/jacs.0c10008
  92. Ryo Nishihara, Kazuki Niwa, Tatsunosuke Tomita, Ryoji Kurita. Coelenterazine Analogue with Human Serum Albumin-Specific Bioluminescence. Bioconjugate Chemistry 2020, 31 (12) , 2679-2684. https://doi.org/10.1021/acs.bioconjchem.0c00536
  93. Hope Adamson, Lars J. C. Jeuken. Engineering Protein Switches for Rapid Diagnostic Tests. ACS Sensors 2020, 5 (10) , 3001-3012. https://doi.org/10.1021/acssensors.0c01831
  94. Emily J. Hanan, Jun Liang, Xiaojing Wang, Robert A. Blake, Nicole Blaquiere, Steven T. Staben. Monomeric Targeted Protein Degraders. Journal of Medicinal Chemistry 2020, 63 (20) , 11330-11361. https://doi.org/10.1021/acs.jmedchem.0c00093
  95. Scott A. Busby, Seth Carbonneau, John Concannon, Christoph E. Dumelin, YounKyoung Lee, Shin Numao, Nicole Renaud, Thomas M. Smith, Douglas S. Auld. Advancements in Assay Technologies and Strategies to Enable Drug Discovery. ACS Chemical Biology 2020, 15 (10) , 2636-2648. https://doi.org/10.1021/acschembio.0c00495
  96. Chee Ka Candice Lam, Kevin Truong. Engineering a Synthesis-Friendly Constitutive Promoter for Mammalian Cell Expression. ACS Synthetic Biology 2020, 9 (10) , 2625-2631. https://doi.org/10.1021/acssynbio.0c00310
  97. Atsushi Ogawa, Yu Itoh. In Vitro Selection of RNA Aptamers Binding to Nanosized DNA for Constructing Artificial Riboswitches. ACS Synthetic Biology 2020, 9 (10) , 2648-2655. https://doi.org/10.1021/acssynbio.0c00384
  98. Hannah S. Zurier, Michelle M. Duong, Julie M. Goddard, Sam R. Nugen. Engineering Biorthogonal Phage-Based Nanobots for Ultrasensitive, In Situ Bacteria Detection. ACS Applied Bio Materials 2020, 3 (9) , 5824-5831. https://doi.org/10.1021/acsabm.0c00546
  99. James M. Wagstaff, Matthew Balmforth, Nick Lewis, Rachel Dods, Catherine Rowland, Katerine van Rietschoten, Liuhong Chen, Helen Harrison, Michael J. Skynner, Michael Dawson, Gabriela Ivanova-Berndt, Paul Beswick. An Assay for Periplasm Entry Advances the Development of Chimeric Peptide Antibiotics. ACS Infectious Diseases 2020, 6 (9) , 2355-2361. https://doi.org/10.1021/acsinfecdis.0c00389
  100. Feng Wang, Zhen-Feng Li, Yuan-Yuan Yang, De-Bin Wan, Natalia Vasylieva, Yu-Qi Zhang, Jun Cai, Hong Wang, Yu-Dong Shen, Zhen-Lin Xu, Bruce D. Hammock. Chemiluminescent Enzyme Immunoassay and Bioluminescent Enzyme Immunoassay for Tenuazonic Acid Mycotoxin by Exploitation of Nanobody and Nanobody–Nanoluciferase Fusion. Analytical Chemistry 2020, 92 (17) , 11935-11942. https://doi.org/10.1021/acs.analchem.0c02338
Load more citations
Download PDF
Go to ACS Chemical Biology
Get e-Alerts
Get e-Alerts

ACS Chemical Biology

Cite this: ACS Chem. Biol. 2012, 7, 11, 1848–1857
Click to copy citationCitation copied!
https://doi.org/10.1021/cb3002478
Published August 15, 2012

Copyright © 2012 American Chemical Society. This publication is licensed under these Terms of Use.

Request reuse permissions

Article Views

51k

Altmetric

-

Citations

1154
Learn about these metrics

Article Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.

Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.

The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated.

  • Abstract

    High Resolution Image
    Download MS PowerPoint Slide

    Figure 1

    Figure 1. Chemical structures. (a) Coelenterazine. (b) Coelenterazine imidazopyrazinone core (with numbering scheme). (c) Furimazine and presumed reaction products.

    High Resolution Image
    Download MS PowerPoint Slide

    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.

    High Resolution Image
    Download MS PowerPoint Slide

    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).

    High Resolution Image
    Download MS PowerPoint Slide

    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.

    High Resolution Image
    Download MS PowerPoint Slide

    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).

    High Resolution Image
    Download MS PowerPoint Slide

    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.

    High Resolution Image
    Download MS PowerPoint Slide

    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.

    High Resolution Image
    Download MS PowerPoint Slide

  • References

    This article references 38 other publications.

    1. 1
      Widder, E. A. (2010) Bioluminescence in the ocean: origins of biological, chemical, and ecological diversity Science 328, 704– 708
      1
      Bioluminescence in the ocean: Origins of biological, chemical, and ecological diversity
      Widder, E. A.
      Science (Washington, DC, United States) (2010), 328 (5979), 704-708CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
      A review. From bacteria to fish, a remarkable variety of marine life depends on bioluminescence (the chem. generation of light) for finding food, attracting mates, and evading predators. Disparate biochem. systems and diverse phylogenetic distribution patterns of light-emitting organisms highlight the ecol. benefits of bioluminescence, with biochem. and genetic analyses providing new insights into the mechanisms of its evolution. The origins and functions of some bioluminescent systems, however, remain obscure. Here, the author reviews recent advances in understanding bioluminescence in the ocean and highlights future research efforts that will unite mol. details with ecol. and evolutionary relations.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXlsFCitbg%253D&md5=c07748c8f13482b1ae8362cec0ffe5e1
    2. 2
      Melnick, J. S., Janes, J., Kim, S., Chang, J. Y., Sipes, D. G., Gunderson, D., Jarnes, L., Matzen, J. T., Garcia, M. E., Hood, T. L., Beigi, R., Xia, G., Harig, R. A., Asatryan, H., Yan, S. F., Zhou, Y., Gu, X. J., Saadat, A., Zhou, V., King, F. J., Shaw, C. M., Su, A. I., Downs, R., Gray, N. S., Schultz, P. G., Warmuth, M., and Caldwell, J. S. (2006) An efficient rapid system for profiling the cellular activities of molecular libraries Proc. Natl. Acad. Sci. U.S.A. 103, 3153– 3158
      There is no corresponding record for this reference.
    3. 3
      Doshi, U. and Li, A. P. (2011) Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction J. Biomol. Screen. 16, 903– 909
      3
      Luciferin IPA-based higher throughput human hepatocyte screening assays for CYP3A4 inhibition and induction
      Doshi, Utkarsh; Li, Albert P.
      Journal of Biomolecular Screening (2011), 16 (8), 903-909CODEN: JBISF3; ISSN:1087-0571. (Sage Publications)
      The authors report here higher throughput screening (HTS) assays for the evaluation of CYP3A4 inhibition and CYP3A4 induction in human hepatocytes using a novel CYP3A4 substrate, luciferin IPA (LIPA). Using human recombinant CYP450 isoforms, LIPA was found to be metabolized extensively by CYP3A4 but not by CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP2E1. In the 384-well plate CYP3A4 inhibition assay, the known inhibitors 1-aminobenzotriazole, erythromycin, ketoconazole, and verapamil were found to cause extensive (max. inhibition of >80%), dose-dependent, statistically significant inhibition of LIPA metab. The non-CYP3A4 inhibitors diethyldithiocarbamate, quercetin, quinidine, sulfaphenazole, ticlopidine, and tranylcypromine were found to have substantially lower (max. inhibition of <50%) or no apparent inhibitory effects in the HTS assay. In the 96-well plate induction assay, the CYP3A4 inducers rifampin, phenobarbital, carbamazepine, phenytoin, troglitazone, rosiglitazone, and pioglitazone yielded dose-dependent induction of LIPA metab., whereas the CYP1A2 inducers omeprazole and 3-methylcholanthrene did not display any induction in the CYP3A4 activity. The high sensitivity and specificity of the assays, the relative ease of execution, and reduced cost, time, and test material requirements suggest that the HTS assays may be applied routinely for screening a large no. of chems. in the drug discovery phase for CYP3A4 inhibitory and inducing potential.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1yitrnM&md5=2533a5fda46939de0805661c6b61a090
    4. 4
      Smirnova, N. A., Haskew-Layton, R. E., Basso, M., Hushpulian, D. M., Payappilly, J. B., Speer, R. E., Ahn, Y. H., Rakhman, I., Cole, P. A., Pinto, J. T., Ratan, R. R., and Gazaryan, I. G. (2011) Development of Neh2-luciferase reporter and its application for high throughput screening and real-time monitoring of Nrf2 activators Chem. Biol. 18, 752– 765
      4
      Development of Neh2-Luciferase Reporter and Its Application for High Throughput Screening and Real-Time Monitoring of Nrf2 Activators
      Smirnova, Natalya A.; Haskew-Layton, Renee E.; Basso, Manuela; Hushpulian, Dmitry M.; Payappilly, Jimmy B.; Speer, Rachel E.; Ahn, Young-Hoon; Rakhman, Ilay; Cole, Philip A.; Pinto, John T.; Ratan, Rajiv R.; Gazaryan, Irina G.
      Chemistry & Biology (Cambridge, MA, United States) (2011), 18 (6), 752-765CODEN: CBOLE2; ISSN:1074-5521. (Cell Press)
      Summary: The NF-E2-related factor 2 (Nrf2) is a key transcriptional regulator of antioxidant defense and detoxification. To directly monitor stabilization of Nrf2, we fused its Neh2 domain, responsible for the interaction with its nucleocytoplasmic regulator, Keap1, to firefly luciferase (Neh2-luciferase). We show that Neh2 domain is sufficient for recognition, ubiquitination, and proteasomal degrdn. of Neh2-luciferase fusion protein. The Neh2-luc reporter system allows direct monitoring of the adaptive response to redox stress and classification of drugs based on the time course of reporter activation. The reporter was used to screen the Spectrum library of 2000 biol. active compds. to identify activators of Nrf2. The most robust and yet nontoxic Nrf2 activators found-nordihydroguaiaretic acid, fisetin, and gedunin-induced astrocyte-dependent neuroprotection from oxidative stress via an Nrf2-dependent mechanism.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXotVejtbc%253D&md5=3919f582cce67396e5b869ce7b9e0325
    5. 5
      Perroy, J., Pontier, S., Charest, P. G., Aubry, M., and Bouvier, M. (2004) Real-time monitoring of ubiquitination in living cells by BRET Nat. Methods 1, 203– 208
      5
      Real-time monitoring of ubiquitination in living cells by BRET
      Perroy, Julie; Pontier, Stephanie; Charest, Pascale G.; Aubry, Muriel; Bouvier, Michel
      Nature Methods (2004), 1 (3), 203-208CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Ubiquitin has emerged as an important regulator of protein stability and function in organisms ranging from yeast to mammals. The ability to detect in situ changes in protein ubiquitination without perturbing the physiol. environment of cells would be a major step forward in understanding the ubiquitination process and its consequences. Here, we describe a new method to study this dynamic post-translational modification in intact human embryonic kidney cells. Using bioLuminescence resonance energy transfer (BRET), we measured the ubiquitination of β-arrestin 2, a regulatory protein implicated in the modulation of G protein-coupled receptors. In addn. to allowing the detection of basal and GPCR-regulated ubiquitination of p-arrestin 2 in living cells, real-time BRET measurements permitted the recording of distinct ubiquitination kinetics that are dictated by the identity of the activated receptor. The ubiquitination BRET assay should prove to be a useful tool for studying the dynamic ubiquitination of proteins and for understanding which cellular functions are regulated by this post-translational event.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXisVGhs74%253D&md5=ae20928d2b7c8208099f526431f89154
    6. 6
      Fan, F., Binkowski, B. F., Butler, B. L., Stecha, P. F., Lewis, M. K., and Wood, K. V. (2008) Novel genetically encoded biosensors using firefly luciferase ACS Chem. Biol. 3, 346– 351
      6
      Novel Genetically Encoded Biosensors Using Firefly Luciferase
      Fan, Frank; Binkowski, Brock F.; Butler, Braeden L.; Stecha, Peter F.; Lewis, Martin K.; Wood, Keith V.
      ACS Chemical Biology (2008), 3 (6), 346-351CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      Genetically encoded biosensors have proven valuable for real-time monitoring of intracellular phenomena, particularly FRET-based sensors incorporating variants of green fluorescent protein. To increase detection sensitivity and response dynamics, the authors genetically engineered firefly luciferase to detect specific intermol. interactions through modulation of its luminescence activity. This concept has been applied in covalent, noncovalent, and allosteric design configurations. The covalent design gives sensitive detection of protease activity through a cleavage-dependent increase in luminescence. The noncovalent and allosteric designs allow reversible detection of the small mols. rapamycin and cAMP, resp. These sensors allow detection of mol. processes within living cells following addn. of the luciferin substrate to the growth medium. For example, the cAMP sensor allows monitoring of intracellular signal transduction assocd. with G-protein coupled receptor function. These and other luminescent biosensors will be useful for the sensitive detection of cellular physiol. in research and drug discovery.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXntlKrsrY%253D&md5=59474ab5133cadb6752eed96e89fe96c
    7. 7
      Remy, I. and Michnick, S. W. (2006) A highly sensitive protein-protein interaction assay based on Gaussia luciferase Nat. Methods 3, 977– 979
      7
      A highly sensitive protein-protein interaction assay based on Gaussia luciferase
      Remy, Ingrid; Michnick, Stephen W.
      Nature Methods (2006), 3 (12), 977-979CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Protein-fragment complementation assays (PCAs) provide a general strategy to study the dynamics of protein-protein interactions in vivo and in vitro. The full potential of PCA requires assays that are fully reversible and sensitive at subendogenous protein expression levels. We describe a new assay that meets these criteria, based on the Gaussia princeps luciferase enzyme, demonstrating chem. reversal, and induction and inhibition of a key interaction linking insulin and TGFβ signaling.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xht1Wqu77I&md5=6d80a3eded2f378506dff827c5af3e04
    8. 8
      Tannous, B. A., Kim, D. E., Fernandez, J. L., Weissleder, R., and Breakefield, X. O. (2005) Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo Mol. Ther. 11, 435– 443
      8
      Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo
      Tannous, Bakhos A.; Kim, Dong-Eog; Fernandez, Juliet L.; Weissleder, Ralph; Breakefield, Xandra O.
      Molecular Therapy (2005), 11 (3), 435-443CODEN: MTOHCK; ISSN:1525-0016. (Elsevier)
      Photoproteins have played a major role in advancing our understanding of biol. processes. A broader array of biocompatible, nontoxic, and novel reporters can serve to expand this potential. Here we describe the properties of a luciferase from the copepod marine organism Gaussia princeps. It is a monomeric protein composed of 185 aa (19.9 kDa) with a short coding sequence (555 bp) making it suitable for viral vectors. The humanized form of Gaussia luciferase (hGLuc) was efficiently expressed in mammalian cells following delivery by HSV-1 amplicon vectors. It was found to be nontoxic and naturally secreted, with flash bioluminescence characteristics similar to those of other coelenterazine luciferases. HGLuc generated over 1000-fold higher bioluminescent signal intensity from live cells together with their immediate environment and over 100-fold higher intensity from viable cells alone (not including secreted luciferase) or cell lysates, compared to humanized forms of firefly (hFLuc) and Renilla (hRLuc) luciferases expressed under similar conditions. Furthermore, hGLuc showed 200-fold higher signal intensity than hRLuc and intensity comparable to that of hFLuc in vivo under std. imaging conditions. Gaussia luciferase provides a sensitive means of imaging gene delivery and other events in living cells in culture and in vivo, with a unique combination of features including high signal intensity, secretion, and ATP independence, thus being able to report from the cells and their environment in real time.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2MXhsVOju7w%253D&md5=bc4ba7b78ea19d52184ebf6415cc410b
    9. 9
      Markova, S. V., Golz, S., Frank, L. A., Kalthof, B., and Vysotski, E. S. (2004) Cloning and expression of cDNA for a luciferase from the marine copepod Metridia longa. A novel secreted bioluminescent reporter enzyme J. Biol. Chem. 279, 3212– 3217
      There is no corresponding record for this reference.
    10. 10
      Nakajima, Y., Kobayashi, K., Yamagishi, K., Enomoto, T., and Ohmiya, Y. (2004) cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca Biosci. Biotechnol. Biochem. 68, 565– 570
      There is no corresponding record for this reference.
    11. 11
      Suzuki, C., Nakajima, Y., Akimoto, H., Wu, C., and Ohmiya, Y. (2005) A new additional reporter enzyme, dinoflagellate luciferase, for monitoring of gene expression in mammalian cells Gene 344, 61– 66
      There is no corresponding record for this reference.
    12. 12
      Inouye, S., Watanabe, K., Nakamura, H., and Shimomura, O. (2000) Secretional luciferase of the luminous shrimp Oplophorus gracilirostris: cDNA cloning of a novel imidazopyrazinone luciferase FEBS Lett. 481, 19– 25
      There is no corresponding record for this reference.
    13. 13
      Wurdinger, T., Badr, C., Pike, L., de Kleine, R., Weissleder, R., Breakefield, X. O., and Tannous, B. A. (2008) A secreted luciferase for ex vivo monitoring of in vivo processes Nat. Methods 5, 171– 173
      13
      A secreted luciferase for ex vivo monitoring of in vivo processes
      Wurdinger, Thomas; Badr, Christian; Pike, Lisa; de Kleine, Ruben; Weissleder, Ralph; Breakefield, Xandra O.; Tannous, Bakhos A.
      Nature Methods (2008), 5 (2), 171-173CODEN: NMAEA3; ISSN:1548-7091. (Nature Publishing Group)
      Luciferases are widely used to monitor biol. processes. Here we describe the naturally secreted Gaussia princeps luciferase (Gluc) as a highly sensitive reporter for quant. assessment of cells in vivo by measuring its concn. in blood. The Gluc blood assay complements in vivo bioluminescence imaging, which has the ability to localize the signal and provides a multifaceted assessment of cell viability, proliferation and location in exptl. disease and therapy models.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXhsV2ksLw%253D&md5=e0cc4226e91256ae0e481f84ab4f3a77
    14. 14
      Andreu, N., Zelmer, A., Fletcher, T., Elkington, P. T., Ward, T. H., Ripoll, J., Parish, T., Bancroft, G. J., Schaible, U., Robertson, B. D., and Wiles, S. (2010) Optimisation of bioluminescent reporters for use with mycobacteria PLoS One 5, e10777
      There is no corresponding record for this reference.
    15. 15
      Shimomura, O., Masugi, T., Johnson, F. H., and Haneda, Y. (1978) Properties and reaction mechanism of the bioluminescence system of the deep-sea shrimp Oplophorus gracilirostris Biochemistry 17, 994– 998
      There is no corresponding record for this reference.
    16. 16
      Kurowski, M. A. and Bujnicki, J. M. (2003) GeneSilico protein structure prediction meta-server Nucleic Acids Res. 31, 3305– 3307
      There is no corresponding record for this reference.
    17. 17
      Flower, D. R., North, A. C., and Sansom, C. E. (2000) The lipocalin protein family: structural and sequence overview Biochim. Biophys. Acta 1482, 9– 24
      There is no corresponding record for this reference.
    18. 18
      Loening, A. M., Fenn, T. D., Wu, A. M., and Gambhir, S. S. (2006) Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output Protein Eng., Des. Sel. 19, 391– 400
      18
      Consensus guided mutagenesis of Renilla luciferase yields enhanced stability and light output
      Loening, Andreas Markus; Fenn, Timothy David; Wu, Anna M.; Gambhir, Sanjiv Sam
      Protein Engineering, Design & Selection (2006), 19 (9), 391-400CODEN: PEDSBR; ISSN:1741-0126. (Oxford University Press)
      Luciferases, which have seen expansive employment as reporter genes in biol. research, could also be used in applications where the protein itself is conjugated to ligands to create probes that are appropriate for use in small animal imaging. As the bioluminescence activity of commonly used luciferases is too labile in serum to permit this application, specific mutations of Renilla luciferase, selected using a consensus sequence driven strategy, were screened for their ability to confer stability of activity in serum as well as their light output. Using this information, a total of eight favorable mutations were combined to generate a mutant Renilla luciferase (RLuc8) that, compared with the parental enzyme, is 200-fold more resistant to inactivation in murine serum and exhibits a 4-fold improvement in light output. Results of the mutational anal. were also used to generate a double mutant optimized for use as a reporter gene. The double mutant had half the resistance to inactivation in serum of the native enzyme while yielding a 5-fold improvement in light output.These variants of Renilla luciferase, which exhibit significantly improved properties compared with the native enzyme, will allow enhanced sensitivity in existing luciferase-based assays as well as enable the development of novel probes labeled with the luciferase protein.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD28Xotl2ksro%253D&md5=c006a4ff7a42c04c1ccf02b80fb1c9ff
    19. 19
      Woo, J. and von Arnim, A. G. (2008) Mutational optimization of the coelenterazine-dependent luciferase from Renilla Plant Methods 4, 23
      There is no corresponding record for this reference.
    20. 20
      Branchini, B. R., Ablamsky, D. M., Davis, A. L., Southworth, T. L., Butler, B., Fan, F., Jathoul, A. P., and Pule, M. A. (2010) Red-emitting luciferases for bioluminescence reporter and imaging applications Anal. Biochem. 396, 290– 297
      There is no corresponding record for this reference.
    21. 21
      Inouye, S. and Shimomura, O. (1997) The use of Renilla luciferase, Oplophorus luciferase, and Apoaequorin as bioluminescent reporter protein in the presence of coelenterazine analogues as substrate Biochem. Biophys. Res. Commun. 233, 349– 353
      There is no corresponding record for this reference.
    22. 22
      Ando, Y., Niwa, K., Yamada, N., Enomoto, T., Irie, T., Kubota, H., Ohmiya, Y., and Akiyama, H. (2008) Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission Nat. Photonics 2, 44– 47
      22
      Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission
      Ando, Yoriko; Niwa, Kazuki; Yamada, Nobuyuki; Enomoto, Toshiteru; Irie, Tsutomu; Kubota, Hidehiro; Ohmiya, Yoshihiro; Akiyama, Hidefumi
      Nature Photonics (2008), 2 (1), 44-47CODEN: NPAHBY; ISSN:1749-4885. (Nature Publishing Group)
      Firefly bioluminescence is the most well-known ideal photoemitter system in biophotonics, known in particular for its extremely high quantum yield, 88 ± 25% (refs. 2,3) or higher, and its magnificent pH-dependent emission-color change between yellow-green and red, modelled as the chem. equil. between two corresponding states. However, the need for re-examn. has also been discussed. In this letter we quantify quantum yields and color changes using our new total-photon-flux spectrometer. We det. the highest quantum yield to be 41.0 ± 7.4% (1 std. deviation (s.d.) est., coverage factor k = 1), and find that bioluminescence spectra are systematically decompd. into one pH-sensitive and two pH-insensitive gaussian components. There is no intensity conversion between yellow-green and red emissions through pH equil., but simple intensity variation of the pH-sensitive gaussian peak at 2.2 eV causes the changes in emission colors. This represents a paradigm shift in the concept of color detn. from long-standing interpretation based on pH equil.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmtVSnuw%253D%253D&md5=00549500c02c8644684b672d7530e863
    23. 23
      Davis, R. E., Zhang, Y. Q., Southall, N., Staudt, L. M., Austin, C. P., Inglese, J., and Auld, D. S. (2007) A cell-based assay for IκBα stabilization using a two-color dual luciferase-based sensor Assay Drug Dev. Technol. 5, 85– 103
      There is no corresponding record for this reference.
    24. 24
      Almond, B., Hawkins, E., Stecha, P., Garvin, D., Paguio, A., Butler, B. L., Beck, M., Wood, M., and Wood, K. (2003) Introducing ChromaLuc technology Promega Notes 85, 11– 14
      There is no corresponding record for this reference.
    25. 25
      Swanson, B., Fan, F., and Wood, K. V. (2007) Enhanced response dynamics for transcription analysis using new pGL4 luciferase reporter vectors Cell Notes 17, 3– 5
      There is no corresponding record for this reference.
    26. 26
      Auld, D. S., Zhang, Y. Q., Southall, N. T., Rai, G., Landsman, M., MacLure, J., Langevin, D., Thomas, C. J., Austin, C. P., and Inglese, J. (2009) A basis for reduced chemical library inhibition of firefly luciferase obtained from directed evolution J. Med. Chem. 52, 1450– 1458
      26
      A Basis for Reduced Chemical Library Inhibition of Firefly Luciferase Obtained from Directed Evolution
      Auld, Douglas S.; Zhang, Ya-Qin; Southall, Noel T.; Rai, Ganesha; Landsman, Marc; MacLure, Jennifer; Langevin, Daniel; Thomas, Craig J.; Austin, Christopher P.; Inglese, James
      Journal of Medicinal Chemistry (2009), 52 (5), 1450-1458CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
      We measured the "druggability" of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo, Promega). Quant. high-throughput screening (qHTS) was used to det. IC50s of 198899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the max. affinity identified in the lucPpy qHTS was 50 nM, while for Kinase-Glo this value increased to 600 nM. Compds. with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compd. interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quant. anal. of structure-activity relationships (>100K compds.) in addressing important questions such as a target's druggability.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhvVWnt7o%253D&md5=aac37056be38a2c0ed06098d2e2f142a
    27. 27
      Fahey, R. C., Hunt, J. S., and Windham, G. C. (1977) On the cysteine and cystine content of proteins. Differences between intracellular and extracellular proteins J. Mol. Evol. 10, 155– 160
      There is no corresponding record for this reference.
    28. 28
      Goerke, A. R., Loening, A. M., Gambhir, S. S., and Swartz, J. R. (2008) Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase Metab. Eng. 10, 187– 200
      28
      Cell-free metabolic engineering promotes high-level production of bioactive Gaussia princeps luciferase
      Goerke, Aaron R.; Loening, Andreas M.; Gambhir, Sanjiv S.; Swartz, James R.
      Metabolic Engineering (2008), 10 (3-4), 187-200CODEN: MEENFM; ISSN:1096-7176. (Elsevier Inc.)
      Due to its small size and intense luminescent signal, Gaussia princeps luciferase (GLuc) is attractive as a potential imaging agent in both cell culture and small animal research models. However, recombinant GLuc prodn. using in vivo techniques has only produced small quantities of active luciferase, likely due to five disulfide bonds being required for full activity. Cell-free biol. provides the freedom to control both the catalyst and chem. compns. in biol. reactions, and we capitalized on this to produce large amts. of highly active GLuc in cell-free reactions. Active yields were improved by mutating the cell ext. source strain to reduce proteolysis, adjusting reaction conditions to enhance oxidative protein folding, further activating energy metab., and encouraging post-translational activation. This cell-free protein synthesis procedure produced 412 μg/mL of purified GLuc, relative to 5 μg/mL isolated for intracellular Escherichia coli expression. The cell-free product had a specific activity of 4.2×1024 photons/s/mol, the highest reported activity for any characterized luciferase.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXnt1ektbc%253D&md5=1f54e550163e57beeae2a131e48a513c
    29. 29
      Inouye, S. and Sasaki, S. (2007) Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris Protein Expression Purif. 56, 261– 268
      29
      Overexpression, purification and characterization of the catalytic component of Oplophorus luciferase in the deep-sea shrimp, Oplophorus gracilirostris
      Inouye, Satoshi; Sasaki, Satoko
      Protein Expression and Purification (2007), 56 (2), 261-268CODEN: PEXPEJ; ISSN:1046-5928. (Elsevier B.V.)
      The luciferase secreted by the deep-sea shrimp Oplophorus consists of 19 and 35 kDa proteins. The 19-kDa protein (19kOLase), the catalytic component of luminescence reaction, was expressed in Escherichia coli using the cold-shock inducted expression system. 19KOLase, expressed as inclusion bodies, was solubilized with 6 M urea and purified by urea-nickel chelate affinity chromatog. The yield of 19kOLase was 16 mg from 400 mL of cultured cells. 19kOLase in 6 M urea could be refolded rapidly by diln. with 50 mM Tris-HCl (pH 7.8)-10 mM EDTA, and the refolded protein showed luminescence activity. The luminescence properties of refolded 19kOLase were characterized, in comparison with native Oplophorus luciferase. Luminescence intensity with bisdeoxycoelenterazine as a substrate was stimulated in the presence of org. solvents. The 19kOLase is a thermolabile protein and is 98 % inhibited by 1 μM Cu2+. The cysteine residue of 19kOLase is not essential for catalysis of the luminescence reaction.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXht1anurrL&md5=0e4fd0d955d54530c24027053fb1cc18
    30. 30
      Wong, G. G., Witek-Giannotti, J., Hewick, R. M., Clark, S. C., and Ogawa, M. (1988) Interleukin 6: identification as a hematopoietic colony-stimulating factor Behring Inst. Mitt. 83, 40– 47
      30
      Interleukin 6: identification as a hematopoietic colony-stimulating factor
      Wong, G. G.; Witek-Giannotti, J.; Hewick, R. M.; Clark, S. C.; Ogawa, M.
      Behring Institute Mitteilungen (1988), 83 (), 40-7CODEN: BHIMA2; ISSN:0301-0457.
      A cDNA encoding a novel myeloid colony-stimulating activity from a human T cell line was identified and mol. cloned. This cytokine proved to be identical to the factor currently known as interleukin (IL)-6, thereby demonstrating effects of IL-6 with hematopoietic target cells. In addn. to its ability to support murine granulocyte-macrophage colony formation, IL-6 acted synergistically with IL-3 in both the murine and human systems in support of colony formation by the primitive blast cell colony forming cell. This multitude of biol. activities suggests that IL-6 plays a prominent role within a network of cytokines in integrating the different arms of the host response to infection.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1MXitFSmur8%253D&md5=b253f52b9e95b28e7ca725a9ef526756
    31. 31
      Lippincott-Schwartz, J., Roberts, T. H., and Hirschberg, K. (2000) Secretory protein trafficking and organelle dynamics in living cells Annu. Rev. Cell Dev. Biol. 16, 557– 589
      31
      Secretory protein trafficking and organelle dynamics in living cells
      Lippincott-Schwartz, Jennifer; Roberts, Theresa H.; Hirschberg, Koret
      Annual Review of Cell and Developmental Biology (2000), 16 (), 557-589CODEN: ARDBF8; ISSN:1081-0706. (Annual Reviews Inc.)
      A review with many refs. Green fluorescent protein chimerae acting as reporters for protein localization and trafficking within the secretory membrane system of living cells have been used in a wide variety of applications, including time-lapse imaging, double-labeling, energy transfer, quantitation, and photobleaching expts. Results from this work are clarifying the steps involved in the formation, translocation, and fusion of transport intermediates; the organization and biogenesis of organelles; and the mechanisms of protein retention, sorting, and recycling in the secretory pathway. In so doing, they are broadening our thinking about the temporal and spatial relationships among secretory organelles and the membrane trafficking pathways that operate between them.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXpvFyq&md5=b5cf93a3d1fd4b750c843c43780bd567
    32. 32
      Simmons, S. O. (2011) Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing Comb. Chem. High Throughput Screening 14, 688– 702
      32
      Fireflies in the coalmine: luciferase technologies in next-generation toxicity testing
      Simmons, Steven O.
      Combinatorial Chemistry & High Throughput Screening (2011), 14 (8), 688-702CODEN: CCHSFU; ISSN:1386-2073. (Bentham Science Publishers Ltd.)
      Whole-animal studies have been the mainstay of toxicity testing for decades. These approaches are too expensive and laborious to effectively characterize all of the chems. currently in com. use. In addn., there are social and ethical pressures to reduce, refine and replace animal testing in toxicol. The National Research Council (NRC) has outlined a new strategy to transition from animal-based tests to high throughput, cell-based assays and computational modeling approaches to characterize chem. toxicants. Crit. to this vision, assays that measure toxicity pathways assocd. with adverse health effects must be developed. Bioluminescent assays are particularly well suited to the demands of next-generation toxicity testing because they measure a wide range of biol. activities in a quant. and high throughput manner. This review describes the limitations of traditional, animal-based toxicity testing and discusses the current and developing uses of bioluminescent technologies in next-generation testing based on three general assays formats: luciferase-limited assays, ATP-limited assays and luciferin-limited assays.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXhtVKqtrnI&md5=1224daf1cf9b892c342b3c87d13c478b
    33. 33
      Rehemtulla, A., Taneja, N., and Ross, B. D. (2004) Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event Mol. Imaging 3, 63– 68
      33
      Bioluminescence detection of cells having stabilized p53 in response to a genotoxic event
      Rehemtulla, Alnawaz; Taneja, Neelam; Ross, Brian D.
      Molecular Imaging (2004), 3 (1), 63-68CODEN: MIOMBP; ISSN:1535-3508. (MIT Press)
      Inactivation of p53 is one of the most frequent mol. events in neoplastic transformation. Approx. 60% of all human tumors have mutations in both p53 alleles. Wild-type p53 activity is regulated in large part by the proteosome-dependent degrdn. of p53, resulting in a short p53 half-life in unstressed and untransformed cells. Activation of p53 by a variety of stimuli, including DNA damage induced by genotoxic drugs or radiation, is accomplished by stabilization of wild-type p53. The stabilized and active p53 can result in either cell-cycle arrest or apoptosis. Surprisingly, the majority of tumor-assocd., inactivating p53 mutations also result in p53 accumulation. Thus, constitutive elevation of p53 levels in cells is a reliable measure of p53 inactivation, whereas transiently increased p53 levels reflect a recent genotoxic stress. In order to facilitate noninvasive imaging of p53 accumulation, the authors here describe the construction of a p53-luciferase fusion protein. Induction of DNA damage in cells expressing, the fusion protein resulted in a time-dependent accumulation of the fusion that was noninvasively detected using bioluminescence imaging and validated by Western blot anal. The p53-Luc protein retains p53 function because its expression in HCT116 cells lacking functional p53 resulted in activation of p21 expression as well as induction of apoptosis in response to a DNA damaging event. Employed in a transgenic animal model, the proposed p53-reporter fusion protein will be useful for studying p53 activation in response to exposure to DNA-damaging carcinogenic agents. It could also be used to study p53 stabilization as a result of inactivating p53 mutations. Such studies will further the understanding of p53's role as the "guardian of the genome" and its function in tumorigenesis.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXkslGjsLc%253D&md5=e3a74f1ffe9966213c680a7668f356e3
    34. 34
      Horn, H. F. and Vousden, K. H. (2007) Coping with stress: multiple ways to activate p53 Oncogene 26, 1306– 1316
      34
      Coping with stress: multiple ways to activate p53
      Horn, H. F.; Vousden, K. H.
      Oncogene (2007), 26 (9), 1306-1316CODEN: ONCNES; ISSN:0950-9232. (Nature Publishing Group)
      A review. Over the years, p53 has been shown to sit at the center of an increasingly complex web of incoming stress signals and outgoing effector pathways. The no. and diversity of stress signals that lead to p53 activation illustrates the breadth of p53's remit - responding to a wide variety of potentially oncogenic insults to prevent tumor development. Interestingly, different stress signals can use different and independent pathways to activate p53, and there is some evidence that different stress signals can mediate different responses. How each of the responses to p53 contributes to inhibition of malignant progression is beginning to be clarified, with the hope that identification of responses that are key to tumor suppression will allow a more focused and effective search for new therapeutic targets. In this review, we will highlight some recently identified roles for p53 in tumor suppression, and discuss some of the numerous mechanisms through which p53 can be regulated and activated.
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2sXitVKhsLY%253D&md5=2d7b83d1eee3c060185f2441868ed16d
    35. 35
      Nishizuka, Y. (1984) The role of protein kinase C in cell surface signal transduction and tumour promotion Nature 308, 693– 698
      35
      The role of protein kinase C in cell surface signal transduction and tumor promotion
      Nishizuka, Yasutomi
      Nature (London, United Kingdom) (1984), 308 (5961), 693-8CODEN: NATUAS; ISSN:0028-0836.
      A review with 80 refs. on the title role of protein kinase C [9026-43-1].
      >> More from SciFinder ®
      https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2cXitVCmtrk%253D&md5=33d139697753d3f7e10417573e131ff9
    36. 36
      Htun, H., Barsony, J., Renyi, I., Gould, D. L., and Hager, G. L. (1996) Visualization of glucocorticoid receptor translocation and intranuclear organization in living cells with a green fluorescent protein chimera Proc. Natl. Acad. Sci. U.S.A. 93, 4845– 4850
      There is no corresponding record for this reference.
    37. 37
      Nakajima, Y., Yamazaki, T., Nishii, S., Noguchi, T., Hoshino, H., Niwa, K., Viviani, V. R., and Ohmiya, Y. (2010) Enhanced beetle luciferase for high-resolution bioluminescence imaging PLoS One 5, e10011
      There is no corresponding record for this reference.
    38. 38
      Suzuki, T., Kondo, C., Kanamori, T., and Inouye, S. (2011) Video rate bioluminescence imaging of secretory proteins in living cells: localization, secretory frequency, and quantification Anal. Biochem. 415, 182– 189
      There is no corresponding record for this reference.

  • Supporting Information

    Supporting Information

    This material is available free of charge via the Internet at http://pubs.acs.org


    Terms & Conditions

    Most electronic Supporting Information files are available without a subscription to ACS Web Editions. Such files may be downloaded by article for research use (if there is a public use license linked to the relevant article, that license may permit other uses). Permission may be obtained from ACS for other uses through requests via the RightsLink permission system: http://pubs.acs.org/page/copyright/permissions.html.