Project description:A dual luciferase reporter (DLR) system utilizing firefly and Renilla luciferases was developed and tested in a model rhizobacterium, Pseudomonas putida KT2440. The DLR was applied to simultaneously analyze expression of three putative bacterioferritin genes (bfralpha, bfrbeta, and bfr) and assess the cellular iron status of strain KT2440 by monitoring expression of the Fur-regulated fepA-fes promoter. The DLR proved to be reproducible and sensitive. Expression of bfralpha (PP0482) and bfrbeta (PP1082) was consistent with expectations for bacterioferritin and varied directly with the iron level. However, expression of bfr (PP4856) was inversely related to the iron concentration and it was thus more likely to encode a Dps-like protein rather than a bacterioferritin.
Project description:We have developed a multiplex reporter system to monitor multiple biological variables in real-time. The secreted Gaussia luciferase was fused to ten different epitope tags (Gluc(tag)), each expressed in different tumor cells. By immunobinding of the tags followed by Gluc(tag) detection, this system allowed the independent and real-time monitoring of mixed cell cultures in vitro and of mixed subcutaneous and intracranial tumor subpopulations in vivo.
Project description:Effective methods for monitoring eukaryotic gene expression and regulation based on bioluminescence - the emission of light by living organisms - are well established. Typically, the expression of a gene of interest is reported on with high sensitivity and over a wide dynamic range by the emission of light from a variety of engineered luciferase genes from beetles and marine organisms. The luciferase reporter genes are expressed downstream of the target gene or promoter and detected after exogenous addition of luciferin substrates. We describe a novel bioluminescence reporter method for the simultaneous monitoring of two genes expressing engineered firefly luciferase variants that emit readily distinguishable green and red light signals. The key feature is the selectivity of the enzymes for two luciferin substrates that determine each emission color. To validate our method, we performed a complex promoter transactivation experiment side-by-side with the Dual-Luciferase Reporter protocol and obtained essentially identical results. Additional comparative experiments demonstrated that our assay system provided improvements in background, cell normalization, and detectability compared to representative available methods. With access to a luminometer equipped with two optical filters, this method is an excellent choice for genetic reporter assays that can be performed with a single reagent solution.
Project description:Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening.
Project description:The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.
Project description:BackgroundProteolysis targeting chimera (PROTAC), a novel drug discovery strategy, utilizes the ubiquitin-proteasome system to degrade target proteins in cells. While Western blotting, mass spectrometry, and Lumit Immunoassay have been instrumental in determining protein levels, the rapid screening of PROTACs continues to pose challenges, necessitating the development of alternative methodologies.ResultsWe herein reported an alternative high-throughput method for screening PROTACs using a dual-reporter system expressing a Renilla luciferase (RLUC)-fused target protein and enhanced green fluorescent protein (EGFP). EGFP served as an internal reference and RLUC as an indicated target protein degradation. Rapid measurement of EGFP or RLUC light signals was achieved using a fluorescence/luminescence plate-based reader in the endpoint mode. The feasibility of the screening model was tested using ARV110, a clinical trial-stage PROTAC targeting the androgen receptor (AR). In EGFP/RLUC-tAR-expressing modal cells treated with varying concentrations of ARV110, normalized RLUC luminescence decreased dose-dependently, as confirmed via western blotting detection of AR expression. Then the platform was used to practically screen Sirtuin 2 (SIRT2) degraders from a small group of PROTACs that we built. Normalized RLUC luminescence changes in model cells expressing EGFP/RLUC-SIRT2 reflected the degradation efficiencies of PROTACs. Compounds 128 and 129 exhibited the highest degradation efficacies, leading to dose-dependent degradation of endogenous SIRT2 protein in the MCF-7 cell line and inducing cell growth arrest.ConclusionsThe dual-reporter system using both fluorescence and chemiluminescence was successfully constructed. Using this method, we identified effective candidate PROTACs against SIRT2. The dual-reporter system may accelerate drug discovery during PROTAC development.
Project description:Studies investigating the action of small RNAs on computationally predicted target genes require some form of experimental validation. Classical molecular methods of validating microRNA action on target genes are laborious, while approaches that tag predicted target sequences to qualitative reporter genes encounter technical limitations. The aim of this study was to address the challenge of experimentally validating large numbers of computationally predicted microRNA-target transcript interactions using an optimized, quantitative, cost-effective, and scalable approach. The presented method combines transient expression via agroinfiltration of Nicotiana benthamiana leaves with a quantitative dual luciferase reporter system, where firefly luciferase is used to report the microRNA-target sequence interaction and Renilla luciferase is used as an internal standard to normalize expression between replicates. We report the appropriate concentration of N. benthamiana leaf extracts and dilution factor to apply in order to avoid inhibition of firefly LUC activity. Furthermore, the optimal ratio of microRNA precursor expression construct to reporter construct and duration of the incubation period post-agroinfiltration were determined. The optimized dual luciferase assay provides an efficient, repeatable and scalable method to validate and quantify microRNA action on predicted target sequences. The optimized assay was used to validate five predicted targets of rice microRNA miR529b, with as few as six technical replicates. The assay can be extended to assess other small RNA-target sequence interactions, including assessing the functionality of an artificial miRNA or an RNAi construct on a targeted sequence.
Project description:Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression.
Project description:BackgroundHuman interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics.ResultsSplit Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner's size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degree C up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system.ConclusionFluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu.
Project description:This study demonstrates the development of a humanized luciferase imaging reporter based on a recently discovered mushroom luciferase (Luz) from Neonothopanus nambi. In vitro and in vivo assessments showed that human-codon-optimized Luz (hLuz) has significantly higher activity than native Luz in various cancer cell types. The potential of hLuz in non-invasive bioluminescence imaging was demonstrated by human tumor xenografts subcutaneously and by the orthotopic lungs xenograft in immunocompromised mice. Luz enzyme or its unique 3OH-hispidin substrate was found to be non-cross-reacting with commonly used luciferase reporters such as Firefly (FLuc2), Renilla (RLuc), or nano-luciferase (NLuc). Based on this feature, a non-overlapping, multiplex luciferase assay using hLuz was envisioned to surpass the limitation of dual reporter assay. Multiplex reporter functionality was demonstrated by designing a new sensor construct to measure the NF-κB transcriptional activity using hLuz and utilized in conjunction with two available constructs, p53-NLuc and PIK3CA promoter-FLuc2. By expressing these constructs in the A2780 cell line, we unveiled a complex macromolecular regulation of high relevance in ovarian cancer. The assays performed elucidated the direct regulatory action of p53 or NF-κB on the PIK3CA promoter. However, only the multiplexed assessment revealed further complexities as stabilized p53 expression attenuates NF-κB transcriptional activity and thereby indirectly influences its regulation on the PIK3CA gene. Thus, this study suggests the importance of live cell multiplexed measurement of gene regulatory function using more than two luciferases to address more realistic situations in disease biology.