Engineered luciferases for molecular sensing in living cells.
ABSTRACT As a means for visualizing molecular physiology within living cells, new strategies are emerging for engineering luciferases into intracellular biosensors. These biosensors can be classified as bimolecular, relying on complementation of luciferase fragments, or intramolecular, relying on domain insertion within the luciferase structure. Multiple design strategies have recently surfaced for the development of intramolecular sensors, allowing dynamic detection of small molecules or post-translational modifications within cells. Building on successes achieved in cell culture, these sensors are now beginning to reveal molecular processes within living organisms.
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ABSTRACT: 2434 Conclusions Methods Subcloning of Caspase-3/7 GloSensor into pEF The bioluminescent reporter was subcloned from Promega's CP vector into pEF by PCR amliication and insertion into MCS at SalI and EcoRI restriction sites. Transfection and clone selection of Caspase -3/7 GloSensor expressing MDA-MB231/1833 cells D54-MG or MDA-MB231/1833 cells were transfected with Caspase-3/7 GloSensor by Fugen reagent following standard protocol and placed in appropriate selection media 48 hrs post transfection. Single clones were selected and tested for reporter expression by western blotting with luciferase antibody (Promega) and for bioluminescence. Clones with similar bioluminescence activity and re-porter expression were selected for experiments. Bioluminescence assay in cell culture Glioma or breast cancer cell lines stably expressing the reporter or diierent reporter versions were seeded in a 96-well assay plate 24 hrs prior to treatment. Cells were treated with 200 ng/ml TRAIL and imaged at indicated timepoints. Live-cell luminescent imaging was performed by adding 100 ug/ml of D-luciferin to the assay media. Photon counts were aquired at diierent timepoints prior-and post-reatment using the Envison luminometer (Perkin Elmer). High throughput screening 1833 reporter cells were seeded in a 96-well plates at a density of 10.000 cells per well by using automated pippetors. 48 hrs post seed-ing media was changed to CO2 independent media containing 1% GloSensor cAMP reagent (ref.1&2)(Promega) and incubated with a compound concentration of 10 uM. Addition of media and compound library was performed using a Titertek Mulidrop Micro-plate Dispensor (Thermo Fisher Scientiic, Watham, MA). 1280 Lopac compound library (Sigma) were used at above indicated concen-trations. Relative Luminescence was calculated by normalizing values of compound treated wells to untreated wells. The Z-factor was calculated as previously described: Zhang et al, Biomol Screen 1999; 4:67-73. Caspase-3/7 GloSensor transgenic mouse generation For the purpose of imaging apoptosis non-invasively in animal models of cancer, we generated a transgenic reporter mouse by pro-nuclear microinjection of a transgene containing the apoptosis reporter (GloSensor Caspase -3/7) into fertilized eggs obtained from FVB/N females. Transgenic mice were generated by the t ransgenic animal model core at the University of Michigan. The schematic in 4 and 5 depicts the transgene. In brief, constitutively active CAG promoter drives expression of EGFP in the absence of Cre ex-pression. A polyA sequence with strong termination signal ensures transcriptional inhibition of the bioluminescent reporter (Caspase-3/7 Glosensor) located 3' of the EGFP cDNA. loxP sites (triangles) are the EGFP cDNA. Cre-mediated excision of the EGFP-stop cassette results in transcription of the bioluminescent reporter (GloSensor caspase-3/7). Establishment of xenograft and GEM models and in vivo bioluminescence assay For breast cancer bone metastasis model 100.000 MDA-MB231/1833 cells stably expressing the Caspase-3/7 GloSensor reporter were implanted into the tibia of the mouse. Tumor growth was followed by MRI and treatment initiated when tumor reached 5-15 mm^3. For in vivo bioluminescence, mice were anasthetized using a 2% isoouorane/air mixture and injected with a single dose of 150 mg/kg D-luciferin itraperitoneally. Consecutive images were acquired before and 6 hrs post treatment or as indicated in by using the IVIS imaging system (Caliper Life Sciences, Hopkinton, MA). Fold induction of bioluminescence activation was calculated by normaliz-ing post treatment values to pre treatment values of each individual animal. Here we describe a new cell death surrogate marker for use in High throughput screening of therapeutics and for in vivo imaging. This reporter, which is based on the split luciferase technology, was evaluated in a breast cancer and glioma cell line and showed a high signal to noise ratio and a wide dynamic range. Robust fold inductions of over 50 fold in vitro to stimuli engaging the receptor mediated or mitochondrial apoptotic pathway were equally translatable to in vivo use. In vivo utility was demonstrated by the use of cancer cell xenografts and the development of a new transgenic mouse model, wherein activation of the Caspase-3/7 GloSensor was achieved by Cre recombination in a tissue dependent manner. The reporter was further found to be highly applicable in HTS and for imaging of cancer cell subpopulations. In summary, we predict that this Caspase biosensor will prove its usefullness in discovering novel drugs and signaling molecules which impinge on Caspase activation in cells and living animals.
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ABSTRACT: Bioluminescent systems are considered as potent reporter systems for bioanalysis since they have specific characteristics, such as relatively high quantum yields and photon emission over a wide range of colors from green to red. Biochemical events are mostly accomplished through large protein machines. These molecular complexes are built from a few to many proteins organized through their interactions. These protein-protein interactions are vital to facilitate the biological activity of cells. The split-luciferase complementation assay makes the study of two or more interacting proteins possible. In this technique, each of the two domains of luciferase is attached to each partner of two interacting proteins. On interaction of those proteins, luciferase fragments are placed close to each other and form a complemented luciferase, which produces a luminescent signal. Split luciferase is an effective tool for assaying biochemical metabolites, where a domain or an intact protein is inserted into an internally fragmented luciferase, resulting in ligand binding, which causes a change in the emitted signals. We review the various applications of this novel luminescent biosensor in studying protein-protein interactions and assaying metabolites involved in analytical biochemistry, cell communication and cell signaling, molecular biology, and the fate of the whole cell, and show that luciferase-based biosensors are powerful tools that can be applied for diagnostic and therapeutic purposes.Analytical and Bioanalytical Chemistry 07/2014; 406(23). · 3.66 Impact Factor
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ABSTRACT: The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.Proceedings of the National Academy of Sciences 10/2014; · 9.81 Impact Factor