Bioluminescent Protein Research Articles

Fluorescence and Bioluminescence Imaging of Angiogenesis in Flk1-Nano-lantern Transgenic Mice

Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.

Real-time analysis of the binding of fluorescent VEGF165a to VEGFR2 in living cells: Effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis. Here we have used a novel stoichiometric protein-labeling method to generate a fluorescent variant of VEGF (VEGF165a-TMR) labeled on a single cysteine within each protomer of the antiparallel VEGF homodimer. VEGF165a-TMR has then been used in conjunction with full length VEGFR2, tagged with the bioluminescent protein NanoLuc, to undertake a real time quantitative evaluation of VEGFR2 binding characteristics in living cells using bioluminescence resonance energy transfer (BRET). This provided quantitative information on VEGF-VEGFR2 interactions. At longer incubation times, VEGFR2 is internalized by VEGF165a-TMR into intracellular endosomes. This internalization can be prevented by the receptor tyrosine kinase inhibitors (RTKIs) cediranib, sorafenib, pazopanib or vandetanib. In the absence of RTKIs, the BRET signal is decreased over time as a consequence of the dissociation of agonist from the receptor in intracellular endosomes and recycling of VEGFR2 back to the plasma membrane.

Bioorthogonal Protein Conjugation: Application for the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon‐γ

Interferon‐γ (IFN‐γ) is a 144‐amino acid long N‐terminal methionylated recombinant protein and is a potent multifunctional cytokine which is secreted primarily by activated NK cells and T cells. Originally came to attention due to its anti‐viral activities, IFN‐γ can also act as an anti‐proliferative, and immunoregulator, as well as a proinflammatory compound. A continuous production of the IFN‐γ along with other inflammatory cytokines is triggered in order to activate the immune system when the virus such as the HIV infects the immune system. Therefore, IFN‐γ is an important biomarker for monitoring infections.The hypothesis for this study is that a bioluminescence‐based immunoassay would provide for highly sensitive detection of IFN‐γ due to high signal‐to‐noise ratio obtained with bioluminescence signal. For this purpose, monoclonal anti‐IFN‐γ antibody was conjugated to a mutant of bioluminescent protein, Gaussia luciferase (GLuc) containing a tyrosine at the N‐terminus. The bioconjugation was achieved through orthogonal chemical conjugation at the N‐terminal tyrosine. It has been demonstrated that the Formylbenzene Diazonium Hexafluorophosphate (FBDP) reagent provides bioorthogonal aldehyde functionality and reacts specifically with the ortho position of the phenol group of the tyrosine residue. The conjugation was efficient and retained the functionalities of both the antibody and the luciferase.A sandwich‐type immunoassay was developed assay and validated for its accuracy and precision. A detection limit of 10−12 M was achieved for the detection of IFN‐γ. We also demonstrated the applicability of the assay for the detection of IFN‐γ in biological fluids such as serum samples.Support or Funding InformationNIGMS funding (R01GM047915)

Abstract P6-01-08: Identifying metastatic drivers in patient derived xenograft models of triple negative breast cancer

Abstract Metastases are responsible for the vast majority of deaths due to breast cancer. Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by high rates of metastasis and poor response to chemotherapy. We are using patient derived xenograft (PDX) models of TNBC to identify drivers of TNBC metastasis. Using these models, we demonstrated that human breast tumors are capable of completing all stages of the metastatic cascade in mice, and metastatic lesions are observed in organs normally found in patients with metastatic breast cancer including lung, liver, bone, brain and lymph nodes. Lentiviral transduction was employed to express both bioluminescent and fluorescent proteins in three distinct PDX models of TNBC. In this way, metastatic lesions can be isolated using bioluminescent imaging and circulating tumor cells (CTCs) are isolated by flow cytometry. A lung metastasis gene expression signature was generated and comprehensive gain-of-function screens are being conducted in vivo to validate this signature and identify functional drivers of TNBC metastasis. Citation Format: Powell E, Shao J, Tieu T, Peoples M, Bristow C, Manyam G, Cai S, Tu Y, Edwards JR, Heffernan TP, Piwnica-Worms D, Liang H, Piwnica-Worms H. Identifying metastatic drivers in patient derived xenograft models of triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-01-08.

Whispering-gallery-mode emission from biological luminescent protein microcavity assemblies

Fluorescence and bioluminescence are widely used to study biological systems from molecular to whole organism level. However, their broadband emission is often a bottleneck for sensitive spectral measurements and multiplexing. To overcome the limitation, the emitters can be coupled with optical cavity modes to generate narrowband spectral features. Here we demonstrate several types of emitter-resonator complexes made of fluorescent or bioluminescent proteins and artificially or naturally formed optical resonators. We engineered cells to express green fluorescent protein (GFP) fused with ABHD5, which binds to oil or lipid droplets supporting whispering gallery modes (WGM). The genetically-integrated complexes feature well-defined WGM spectral peaks. We measured WGM peaks from GFP-coated BaTiO3 beads (2.56 μm in diameter) during mitosis. Finally, we demonstrate cavity-enhanced bioluminescence using luciferase-coated beads and biochemical excitation. The ability to tailor spontaneous emission by cavity resonance inside biological systems should have applications in biological sensing, imaging and cell tagging.

Expression of a soluble truncated Vargula luciferase in Escherichia coli

Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.

Tyr72 and Tyr80 are Involved in the Formation of an Active Site of a Luciferase of Copepod Metridia longa.

Luciferase of copepod Metridia longa (MLuc) is a naturally secreted enzyme catalyzing the oxidative decarboxylation of coelenterazine with the emission of light. To date, three nonallelic isoforms of different lengths (17-24 kDa) for M. longa luciferase have been cloned. All the isoforms are single-chain proteins consisting of a 17-residue signal peptide for secretion, variable N-terminal part and conservative C-terminus responsible for luciferase activity. In contrast to other bioluminescent proteins containing a lot of aromatic residues which are frequently involved in light emission reaction, the C-terminal part of MLuc contains only four Phe, two Tyr, one Trp and two His residues. To figure out whether Tyr residues influence bioluminescence, we constructed the mutants with substitution of Tyr to Phe (Y72F and Y80F). Tyrosine substitutions do not eliminate the ability of luciferase to bioluminescence albeit significantly reduce relative specific activity and change bioluminescence kinetics. In addition, the Tyr replacements have no effect on bioluminescence spectrum, thereby indicating that tyrosines are not involved in the emitter formation. However, as it was found that the intrinsic fluorescence caused by Tyr residues is quenched by a reaction substrate, coelenterazine, in concentration-dependent manner, we infer that both tyrosine residues are located in the luciferase substrate-binding cavity.

Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids.

Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.

Concurrent Modulation of Quantum Dot Photoluminescence Using a Combination of Charge Transfer and Förster Resonance Energy Transfer: Competitive Quenching and Multiplexed Biosensing Modality.

An emerging trend with semiconductor quantum dots (QDs) is their use as scaffolds to assemble multiple energy transfer pathways. Examples to date have combined various competitive and sequential Förster resonance energy transfer (FRET) pathways between QDs and fluorescent dyes, luminescent lanthanide complexes, and bioluminescent proteins. Here, we show that the photoluminescence (PL) of QD bioconjugates can also be modulated by a combination of FRET and charge transfer (CT), and characterize the concurrent effects of these mechanistically different pathways using PL measurements at both the ensemble and the single particle level. Peptides were distally labeled with either a fluorescent dye that quenched QD PL through FRET or a ruthenium(II) phenanthroline complex that quenched QD PL through electron transfer. The labeled peptides were assembled around a central CdSe/ZnS QD at different ratios, tuning the relative rates of FRET and CT, which were competitive quenching pathways. The concurrent effects of FRET and CT were predictable from a rate analysis that was calibrated to the isolated effects of each of these pathways. Notably, the dye/QD PL intensity ratio reflected changes in the relative rate of FRET but was approximately independent of CT. In turn, the sum of the QD and dye PL intensities, when adjusted for quantum yields, reflected changes in the relative rate of CT quenching, approximately independent of FRET. The capacity for multiplexed sensing of protease activity was demonstrated using these two orthogonal detection channels. Combined CT-FRET configurations with QDs are thus promising for applications in bioanalysis, sensing, and imaging, and may prove useful in other photonic applications.

Exploiting NanoLuc luciferase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity.

The availability of smartphones with high-performance digital image sensors and processing power has completely reshaped the landscape of point-of-need analysis. Thanks to the high maturity level of reporter gene technology and the availability of several bioluminescent proteins with improved features, we were able to develop a bioluminescence smartphone-based biosensing platform exploiting the highly sensitive NanoLuc luciferase as reporter. A 3D-printed smartphone-integrated cell biosensor based on genetically engineered Hek293T cells was developed. Quantitative assessment of (anti)-inflammatory activity and toxicity of liquid samples was performed with a simple and rapid add-and-measure procedure. White grape pomace extracts, known to contain several bioactive compounds, were analyzed, confirming the suitability of the smartphone biosensing platform for analysis of untreated complex biological matrices. Such approach could meet the needs of small medium enterprises lacking fully equipped laboratories for first-level safety tests and rapid screening of new bioactive products. Graphical abstract Smartphone-based bioluminescence cell biosensor.

Near-infrared bioluminescent proteins for two-color multimodal imaging.

Bioluminescence imaging became a widely used technique for noninvasive study of biological processes in small animals. Bioluminescent probes with emission in near-infrared (NIR) spectral region confer the advantage of having deep tissue penetration capacity. However, there are a very limited number of currently available luciferases that exhibit NIR bioluminescence. Here, we engineered two novel chimeric probes based on RLuc8 luciferase fused with iRFP670 and iRFP720 NIR fluorescent proteins. Due to an intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, the chimeric luciferases exhibit NIR bioluminescence with maxima at 670 nm and 720 nm, respectively. The 50 nm spectral shift between emissions of the two iRFP chimeras enables combined multicolor bioluminescence imaging (BLI) and the respective multicolor fluorescence imaging (FLI) of the iRFPs. We show that for subcutaneously implanted cells, NIR bioluminescence provided a 10-fold increase in sensitivity compared to NIR FLI. In deep tissues, NIR BLI enabled detection of as low as 104 cells. Both BLI and FLI allowed monitoring of tumor growth and metastasis from early to late stages. Multimodal imaging, which combines concurrent BLI and FLI, provides continuous spatiotemporal analysis of metastatic cells in animals, including their localization and quantification.

Lentiviral transduction and subsequent loading with nanoparticles do not affect cell viability and proliferation in hair-follicle-bulge-derived stem cells in vitro.

The application of stem cells in the treatment of various degenerative diseases is highly promising. However, cell-based therapy could be limited by the problem of low viability of grafted cells and uncertainty about their fate. The combination of molecular imaging and contrast-enhanced MRI may give more insight into the survival and behavior of grafted stem cells. We explore hair-follicle-bulge-derived stem cells (HFBSCs) as a potential candidate for autologous cell-based therapy. HFBSCs are transduced with a lentiviral construct with genes coding for bioluminescent (Luc2) and fluorescent (copGFP) reporter proteins, and subsequently loaded with magnetic nanoparticles to enable MRI visualization. Thus, we investigate for the first time if lentiviral transduction and cellular loading with nanoparticles have a cytotoxic effect upon these stem cells. Transduction efficiency, proliferation rate, cell viability and reporter protein co-expression during long-term culture of transduced HFBSCs were studied using fluorescence and bioluminescence microscopy. In addition, the effect of TMSR50 nanoparticles on proliferation and viability was investigated using the MTS assay and bioluminescence microscopy. The amount of TMSR50-loaded HFBSCs needed to reach signal threshold for MRI was assessed using an agarose phantom. Transduction with the Luc2-copGFP construct did not influence senescence, proliferation, doubling time, and differentiation of the HFBSCs. CopGFP expression was visible immediately after transduction and persisted for at least 15 passages, concomitantly with Luc2 expression. Cellular loading with TMSR50 nanoparticles did not affect cell viability and proliferation. The results imply that combined MRI and bioluminescence imaging may enable in vivo localization and long-term monitoring of grafted viable HFBSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Bioluminescent jellyfish proteins used to produce low-energy laser

Bioluminescent jellyfish proteins used to produce low-energy laser

Fluorescent and Bioluminescent Reporter Myxoviruses.

The advent of virus reverse genetics has enabled the incorporation of genetically encoded reporter proteins into replication-competent viruses. These reporters include fluorescent proteins which have intrinsic chromophores that absorb light and re-emit it at lower wavelengths, and bioluminescent proteins which are luciferase enzymes that react with substrates to produce visible light. The incorporation of these reporters into replication-competent viruses has revolutionized our understanding of molecular virology and aspects of viral tropism and transmission. Reporter viruses have also enabled the development of high-throughput assays to screen antiviral compounds and antibodies and to perform neutralization assays. However, there remain technical challenges with the design of replication-competent reporter viruses, and each reporter has unique advantages and disadvantages for specific applications. This review describes currently available reporters, design strategies for incorporating reporters into replication-competent paramyxoviruses and orthomyxoviruses, and the variety of applications for which these tools can be utilized both in vitro and in vivo.

A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo.

Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals due to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright engineered orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter in vivo, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.

NanoLuc: A Small Luciferase Is Brightening Up the Field of Bioluminescence.

The biomedical field has greatly benefited from the discovery of bioluminescent proteins. Currently, scientists employ bioluminescent systems for numerous biomedical applications, ranging from highly sensitive cellular assays to bioluminescence-based molecular imaging. Traditionally, these systems are based on Firefly and Renilla luciferases; however, the applicability of these enzymes is limited by their size, stability, and luminescence efficiency. NanoLuc (NLuc), a novel bioluminescence platform, offers several advantages over established systems, including enhanced stability, smaller size, and >150-fold increase in luminescence. In addition, the substrate for NLuc displays enhanced stability and lower background activity, opening up new possibilities in the field of bioluminescence imaging. The NLuc system is incredibly versatile and may be utilized for a wide array of applications. The increased sensitivity, high stability, and small size of the NLuc system have the potential to drastically change the field of reporter assays in the future. However, as with all such technology, NLuc has limitations (including a nonideal emission for in vivo applications and its unique substrate) which may cause it to find restricted use in certain areas of molecular biology. As this unique technology continues to broaden, NLuc may have a significant impact in both preclinical and clinical fields, with potential roles in disease detection, molecular imaging, and therapeutic monitoring. This review will present the NLuc technology to the scientific community in a nonbiased manner, allowing the audience to adopt their own views of this novel system.

Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.

Light dazzles from the black box: whole-cell biosensors are ready to inform on fundamental soil biological processes

Whole-cell biosensors are natural or engineered microorganisms producing signals in response to specific stimuli. This review introduces the use of whole-cell biosensors for the study of the soil system, discuss the recent developments and some current limitations and draws future prospects of the whole-cell biosensors for application to the study of the agro-ecosystems. The review focuses mainly on the lux- and gfp-inserted whole-cell biosensors producing bioluminescence and multicoloured fluorescent proteins, which allow an easy and reproducible detection of the signals from a large number of prokaryotic and eukaryotic soil-borne microorganisms. This review also points out how the whole-cell biosensors indicate the bioavailability of selected analyte, an information that cannot be straight forwardly extrapolated using the chemical methods of soil analysis. However, regardless of the immense progress in biotechnology and genetics that allows to construct whole-cell biosensors for virtually detecting any chemical at ultra low concentrations, the soil still remains the most extreme natural system to be studied with these biotechnological analytical tools. Although a lack of standardization for most of the constructed whole-cell biosensors along with the scarce knowledge of their performance concur to prevent their use in the official methods of soil and environmental analysis, owing to their stability and selectivity we restate that the whole-cell biosensors are ready to provide information on the main processes occurring in soil, and represent unprecedented sensitive tools for improving agriculture and for soil monitoring.

NanoLuc Luciferase - A Multifunctional Tool for High Throughput Antibody Screening.

Based on the recent development of NanoLuc luciferase (Nluc), a small (19 kDa), highly stable, ATP independent, bioluminescent protein, an extremely robust and ultra high sensitivity screening system has been developed whereby primary hits of therapeutic antibodies and antibody fragments could be characterized and quantified without purification. This system is very versatile allowing cellular and solid phase ELISA but also homogeneous BRET based screening assays, relative affinity determinations with competition ELISA and direct Western blotting. The new Nluc protein fusion represents a “swiss army knife solution” for today and future high throughput antibody drug screenings.

A Rapid, Semi-Quantitative Assay to Screen for Modulators of Alpha-Synuclein Oligomerization Ex vivo.

Alpha synuclein (αsyn) aggregates are associated with the pathogenesis of Parkinson's disease and others related disorders. Although modulation of αsyn aggregation is an attractive therapeutic target, new powerful methodologies are desperately needed to facilitate in vivo screening of novel therapeutics. Here, we describe an in vivo rodent model with the unique ability to rapidly track αsyn-αsyn interactions and thus oligomerization using a bioluminescent protein complementation strategy that monitors spatial and temporal αsyn oligomerization ex vivo. We find that αsyn forms oligomers in vivo as early as 1 week after stereotactic AAV injection into rat substantia nigra. Strikingly, although abundant αsyn expression is also detected in striatum at 1 week, no αsyn oligomers are detected at this time point. By 4 weeks, oligomerization of αsyn is detected in both striatum and substantia nigra homogenates. Moreover, in a proof-of-principle experiment, the effect of a previously described Hsp90 inhibitor known to prevent αsyn oligomer formation, demonstrates the utility of this rapid and sensitive animal model to monitor αsyn oligomerization status in the rat brain.