Journal articles: 'Luciferasi'

1

Heise, Kerstin, Henry Oppermann, Jürgen Meixensberger, Rolf Gebhardt, and Frank Gaunitz. "Dual Luciferase Assay for Secreted Luciferases Based onGaussiaand NanoLuc." ASSAY and Drug Development Technologies 11, no. 4 (2013): 244–52. http://dx.doi.org/10.1089/adt.2013.509.

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Delroisse, Jérôme, Esther Ullrich-Lüter, Stefanie Blaue, et al. "A puzzling homology: a brittle star using a putative cnidarian-type luciferase for bioluminescence." Open Biology 7, no. 4 (2017): 160300. http://dx.doi.org/10.1098/rsob.160300.

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Bioluminescence relies on the oxidation of a luciferin substrate catalysed by a luciferase enzyme. Luciferins and luciferases are generic terms used to describe a large variety of substrates and enzymes. Whereas luciferins can be shared by phylogenetically distant organisms which feed on organisms producing them, luciferases have been thought to be lineage-specific enzymes. Numerous light emission systems would then have co-emerged independently along the tree of life resulting in a plethora of non-homologous luciferases. Here, we identify for the first time a candidate luciferase of a luminous echinoderm, the ophiuroid Amphiura filiformis . Phylogenomic analyses identified the brittle star predicted luciferase as homologous to the luciferase of the sea pansy Renilla (Cnidaria), contradicting with the traditional viewpoint according to which luciferases would generally be of convergent origins. The similarity between the Renilla and Amphiura luciferases allowed us to detect the latter using anti- Renilla luciferase antibodies. Luciferase expression was specifically localized in the spines which were demonstrated to be the bioluminescent organs in vivo . However, enzymes homologous to the Renilla luciferase but unable to trigger light emission were also identified in non-luminous echinoderms and metazoans. Our findings strongly indicate that those enzymes, belonging to the haloalkane dehalogenase family, might then have been convergently co-opted into luciferases in cnidarians and echinoderms. In these two benthic suspension-feeding species, similar ecological pressures would constitute strong selective forces for the functional shift of these enzymes and the emergence of bioluminescence.

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HOSSEINKHANI, Saman, Rose SZITTNER, and Edward A. MEIGHEN. "Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay." Biochemical Journal 385, no. 2 (2005): 575–80. http://dx.doi.org/10.1042/bj20040863.

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Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the ‘slow’ X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820–13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175→Gly)] resulted in the ‘slow decay’ X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between ‘slow’ and ‘fast’ luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase–flavin–oxygen intermediate.

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Viviani, Vadim R., Gabriel F. Pelentir, and Vanessa R. Bevilaqua. "Bioluminescence Color-Tuning Firefly Luciferases: Engineering and Prospects for Real-Time Intracellular pH Imaging and Heavy Metal Biosensing." Biosensors 12, no. 6 (2022): 400. http://dx.doi.org/10.3390/bios12060400.

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Firefly luciferases catalyze the efficient production of yellow-green light under normal physiological conditions, having been extensively used for bioanalytical purposes for over 5 decades. Under acidic conditions, high temperatures and the presence of heavy metals, they produce red light, a property that is called pH-sensitivity or pH-dependency. Despite the demand for physiological intracellular biosensors for pH and heavy metals, firefly luciferase pH and metal sensitivities were considered drawbacks in analytical assays. We first demonstrated that firefly luciferases and their pH and metal sensitivities can be harnessed to estimate intracellular pH variations and toxic metal concentrations through ratiometric analysis. Using Macrolampis sp2 firefly luciferase, the intracellular pH could be ratiometrically estimated in bacteria and then in mammalian cells. The luciferases of Macrolampis sp2 and Cratomorphus distinctus fireflies were also harnessed to ratiometrically estimate zinc, mercury and other toxic metal concentrations in the micromolar range. The temperature was also ratiometrically estimated using firefly luciferases. The identification and engineering of metal-binding sites have allowed the development of novel luciferases that are more specific to certain metals. The luciferase of the Amydetes viviani firefly was selected for its special sensitivity to cadmium and mercury, and for its stability at higher temperatures. These color-tuning luciferases can potentially be used with smartphones for hands-on field analysis of water contamination and biochemistry teaching assays. Thus, firefly luciferases are novel color-tuning sensors for intracellular pH and toxic metals. Furthermore, a single luciferase gene is potentially useful as a dual bioluminescent reporter to simultaneously report intracellular ATP and/or luciferase concentrations luminometrically, and pH or metal concentrations ratiometrically, providing a useful tool for real-time imaging of intracellular dynamics and stress.

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Tafreshi, Narges Kh, Majid Sadeghizadeh, Rahman Emamzadeh, Bijan Ranjbar, Hossein Naderi-Manesh, and Saman Hosseinkhani. "Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases." Biochemical Journal 412, no. 1 (2008): 27–33. http://dx.doi.org/10.1042/bj20070733.

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The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as Km and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

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Kim, Sung Bae, Ryo Nishihara, Daniel Citterio, and Koji Suzuki. "Fabrication of a New Lineage of Artificial Luciferases from Natural Luciferase Pools." ACS Combinatorial Science 19, no. 9 (2017): 594–99. http://dx.doi.org/10.1021/acscombsci.7b00081.

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Kotlobay, A. A., Z. M. Kaskova, and I. V. Yampolsky. "Palette of Luciferases: Natural Biotools for New Applications in Biomedicine." Acta Naturae 12, no. 2 (2020): 15–27. http://dx.doi.org/10.32607/actanaturae.10967.

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Optoanalytical methods based on using genetically encoded bioluminescent enzymes,luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal.The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Amongseveral naturally available luciferases, a few have been found to be useful for practicalapplication. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors areparameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based ontheir biochemical properties before choosing one bioluminescent probe suitable for a specific application.

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Kotlobay, A. A., Z. M. Kaskova, and I. V. Yampolsky. "Palette of Luciferases: Natural Biotools for New Applications in Biomedicine." Acta Naturae 12, no. 2 (2020): 15–27. http://dx.doi.org/10.32607/actanaturae.11152.

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Optoanalytical methods based on using genetically encoded bioluminescent enzymes,luciferases, allow one to obtain highly sensitive signals, are non-invasive, and require no external irradiation. Bioluminescence is based on the chemical reaction of oxidation of a low-molecular-weight substrate (luciferin) by atmospheric oxygen, which is catalyzed by an enzyme (luciferase). Relaxation of the luciferin oxidation product from its excited state is accompanied by a release of a quantum of light, which can be detected as an analytical signal.The ability to express luciferase genes in various heterological systems and high quantum yields of luminescence reactions have made these tools rather popular in biology and medicine. Amongseveral naturally available luciferases, a few have been found to be useful for practicalapplication. Luciferase size, the wavelength of its luminescence maximum, enzyme thermostability, optimal pH of the reaction, and the need for cofactors areparameters that may differ for luciferases from different groups of organisms, and this fact directly affects the choice of the application area for each enzyme. It is quite important to overview the whole range of currently available luciferases based ontheir biochemical properties before choosing one bioluminescent probe suitable for a specific application.

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SALA-NEWBY, Graciela B., Catherine M. THOMSON, and Anthony K. CAMPBELL. "Sequence and biochemical similarities between the luciferases of the glow-worm Lampyris noctiluca and the firefly Photinus pyralis." Biochemical Journal 313, no. 3 (1996): 761–67. http://dx.doi.org/10.1042/bj3130761.

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A full-length clone encoding Lampyris noctiluca (British glow-worm) luciferase was isolated from a complementary DNA (cDNA) expression library constructed with mRNA extracted from light organs. The luciferase was a 547-residue protein, as deduced from the nucleotide sequence. The protein was closely related to those of other lampyrid beetles, the similarity to Photinus pyralis luciferase being 84% and to Luciola 67%. In contrast, Lampyris luciferase had less sequence similarity to the luciferases of the click beetle Pyrophorus, at 48%. Engineering Lampyris luciferase in vitro showed that the C-terminal peptide containing 12 amino acids in Photinus and 9 amino acids in Lampyris was essential for bioluminescence. The pH optimum and the Km values for ATP and luciferin were similar for both Photinus and Lampyris luciferases, although the light emitted by the latter shifted towards the blue and was less stable at 37 °C. It was concluded that the molecular and biochemical properties were not sufficient to explain the glowing or flashing of the two beetles Lampyris and Photinus.

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Carrasco-López, César, Juliana C. Ferreira, Nathan M. Lui, et al. "Beetle luciferases with naturally red- and blue-shifted emission." Life Science Alliance 1, no. 4 (2018): e201800072. http://dx.doi.org/10.26508/lsa.201800072.

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The different colors of light emitted by bioluminescent beetles that use an identical substrate and chemiexcitation reaction sequence to generate light remain a challenging and controversial mechanistic conundrum. The crystal structures of two beetle luciferases with red- and blue-shifted light relative to the green yellow light of the common firefly species provide direct insight into the molecular origin of the bioluminescence color. The structure of a blue-shifted green-emitting luciferase from the firefly Amydetes vivianii is monomeric with a structural fold similar to the previously reported firefly luciferases. The only known naturally red-emitting luciferase from the glow-worm Phrixothrix hirtus exists as tetramers and octamers. Structural and computational analyses reveal varying aperture between the two domains enclosing the active site. Mutagenesis analysis identified two conserved loops that contribute to the color of the emitted light. These results are expected to advance comparative computational studies into the conformational landscape of the luciferase reaction sequence.

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Kamiya, Genta, Nobuo Kitada, Tadaomi Furuta, Takashi Hirano, Shojiro Maki, and Sung Bae Kim. "C-Series Coelenterazine-Driven Bioluminescence Signature Imaging." International Journal of Molecular Sciences 23, no. 21 (2022): 13047. http://dx.doi.org/10.3390/ijms232113047.

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The present study introduces a unique BL signature imaging system with novel CTZ analogues named “C-series.” Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases.

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Amaral, Danilo T., Gabriela Oliveira, Jaqueline R. Silva, and Vadim R. Viviani. "A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations." Photochemical & Photobiological Sciences 15, no. 9 (2016): 1148–54. http://dx.doi.org/10.1039/c6pp00165c.

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The new orange bioluminescence eliciting luciferase from the Amazon P. angustus click-beetle was cloned and sequenced, showing that the substitutions at position 247 is critical for modulating bioluminescence colors in click beetle luciferases.

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Chini, A., S. Murray, J. Grant, C. Thomson, and G. Loake. "Characterisation of a PR-1: Luciferase transgenic line deployed to uncover novel defence-related Arabidopsis mutants by luciferase imaging." Plant Protection Science 38, SI 2 - 6th Conf EFPP 2002 (2017): 615–16. http://dx.doi.org/10.17221/10570-pps.

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In order to identify components of the defence signalling network that may contribute to the establishment of disease resistance, we generated a novel PR-1::Luciferase transgenic line which was deployed in an imaging based screen to uncover novel defence-related mutants. Approximately, 5000 ethylmethane sulfonate (EMS) lines and 30 000 activation tagged lines were generated and screened for enhanced LUC activity via ultra low light imaging.

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Braeuning, Albert, та Silvia Vetter. "The nuclear factor κB inhibitor (E)-2-fluoro-4′-methoxystilbene inhibits firefly luciferase". Bioscience Reports 32, № 6 (2012): 531–37. http://dx.doi.org/10.1042/bsr20120043.

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Photinus pyralis (firefly) luciferase is widely used as a reporter system to monitor alterations in gene promoter and/or signalling pathway activities in vitro. The enzyme catalyses the formation of oxyluciferin from D-luciferin in an ATP-consuming reaction involving photon emission. The purpose of the present study was to characterize the luciferase-inhibiting potential of (E)-2-fluoro-4′-methoxystilbene, which is known as a potent inhibitor of the NF-κB (nuclear factor κB) signalling pathway that is used to modulate the NF-κB signalling pathway in vitro. Results show that (E)-2-fluoro-4′-methoxystilbene effectively inhibits firefly luciferase activity in cell lysates and living cells in a non-competitive manner with respect to the luciferase substrates D-luciferin and ATP. By contrast, the compound has no effect on Renilla and Gaussia luciferases. The mechanism of firefly luciferase inhibition by (E)-2-fluoro-4′-methoxystilbene, as well as its potency is comparable to its structure analogue resveratrol. The in vitro use of trans-stilbenes such as (E)-2-fluoro-4′-methoxystilbene or resveratrol compromises firefly luciferase reporter assays as well as ATP/luciferase-based cell viability assays.

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Kenda, Maša, Jan Vegelj, Barbara Herlah, Andrej Perdih, Přemysl Mladěnka, and Marija Sollner Dolenc. "Evaluation of Firefly and Renilla Luciferase Inhibition in Reporter-Gene Assays: A Case of Isoflavonoids." International Journal of Molecular Sciences 22, no. 13 (2021): 6927. http://dx.doi.org/10.3390/ijms22136927.

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Firefly luciferase is susceptible to inhibition and stabilization by compounds under investigation for biological activity and toxicity. This can lead to false-positive results in in vitro cell-based assays. However, firefly luciferase remains one of the most commonly used reporter genes. Here, we evaluated isoflavonoids for inhibition of firefly luciferase. These natural compounds are often studied using luciferase reporter-gene assays. We used a quantitative structure–activity relationship (QSAR) model to compare the results of in silico predictions with a newly developed in vitro assay that enables concomitant detection of inhibition of firefly and Renilla luciferases. The QSAR model predicted a moderate to high likelihood of firefly luciferase inhibition for all of the 11 isoflavonoids investigated, and the in vitro assays confirmed this for seven of them: daidzein, genistein, glycitein, prunetin, biochanin A, calycosin, and formononetin. In contrast, none of the 11 isoflavonoids inhibited Renilla luciferase. Molecular docking calculations indicated that isoflavonoids interact favorably with the D-luciferin binding pocket of firefly luciferase. These data demonstrate the importance of reporter-enzyme inhibition when studying the effects of such compounds and suggest that this in vitro assay can be used to exclude false-positives due to firefly or Renilla luciferase inhibition, and to thus define the most appropriate reporter gene.

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Kargar, Farzane, Mojtaba Mortazavi, Masoud Torkzadeh-Mahani, Safa Lotfi, and Shahryar Shakeri. "Evaluation of Luciferase Thermal Stability by Arginine Saturation in the Flexible Loops." Current Proteomics 17, no. 1 (2020): 30–39. http://dx.doi.org/10.2174/1570164616666190320151005.

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Background: The firefly luciferase enzyme is widely used in protein engineering and diverse areas of biotechnology, but the main problem with this enzyme is low-temperature stability. Previous reports indicated that surface areas of thermostable proteins are rich in arginine, which increased their thermal stability. In this study, this aspect of thermophilic proteins evaluated by mutations of surface residues to Arg. Here, we report the construction, purification, and studying of these mutated luciferases. Methods: For mutagenesis, the QuikChange site-directed mutagenesis was used and the I108R, T156R, and N177R mutant luciferases were created. In the following, the expression and purification of wild-type and mutant luciferases were conducted and their kinetic and structural properties were analyzed. To analyze the role of these Arg in these loops, the 3D models of these mutants’ enzymes were constructed in the I-TASSER server and the exact situation of these mutants was studied by the SPDBV and PyMOL software. Results: Overall, the optimum temperature of these mutated enzymes was not changed. However, after 30 min incubation of these mutated enzymes at 30°C, the I108R, T156R, N177R, and wild-type kept the 80%, 50%, 20%, and 20% of their original activity, respectively. It should be noted that substitution of these residues by Arg preserved the specific activity of firefly luciferase. Conclusion: Based on these results, it can be concluded that T156R and N177R mutants by compacting local protein structure, increased the thermostability of luciferase. However, insertion of positively charged residues in these positions create the new hydrogen bonds that associated with a series of structural changes and confirmed by intrinsic and extrinsic fluorescence spectroscopy and homology modeling studies.

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Viviani, Vadim R., Vanessa R. Bevilaqua, Daniel R. de Souza, Gabriel F. Pelentir, Michio Kakiuchi, and Takashi Hirano. "A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6′-Amino-Analogs for Bioimaging Purposes." International Journal of Molecular Sciences 22, no. 1 (2020): 303. http://dx.doi.org/10.3390/ijms22010303.

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Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6′-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636–655 nm. Among them, the combination of PxRE-R215K mutant with 6′-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

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Nemtseva, Elena V., Dmitry V. Gulnov, Marina A. Gerasimova, et al. "Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy." International Journal of Molecular Sciences 22, no. 19 (2021): 10449. http://dx.doi.org/10.3390/ijms221910449.

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Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

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AMIDON, Wendy J., Joanne E. PFEIL, and Susannah GAL. "Modification of luciferase to be a substrate for plant aspartic proteinase." Biochemical Journal 343, no. 2 (1999): 425–33. http://dx.doi.org/10.1042/bj3430425.

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The possibility of using firefly luciferase as a substrate for an aspartic proteinase was explored. Several amino acid modifications to the C-terminus of the luciferase were created on the basis of the known substrate of the Arabidopsis thaliana aspartic proteinase, pro-(barley lectin). One luciferase with the sequence Arg-Asp-Gly-Val-Phe-Ala-Ala instead of the native Arg-Glu-Ile-Leu-Ile-Lys-Ala at position -15 to -9 relative to the C-terminus of native luciferase was found to possess 17% of the original luciferase activity. When this modified luciferase was incubated with the aspartic proteinase, a specific loss in activity occurred that was not observed with the original luciferase. However, both enzymes seemed very sensitive to the acidic conditions required for aspartic proteinase activity. The other versions of luciferase with different numbers of pro-(barley lectin) amino acids were not active luciferases. This provided information on the structural requirements of the C-terminal portion of the protein for luciferase activity. The luciferase proteins were also monitored during the digestion by using Western blots and some were shown to be substrates for the aspartic proteinase. Contrary to what had been expected, the modified luciferase that incorporated the pro-(barley lectin) sequences was not simply cleaved at the engineered site but at additional positions in the protein. The Arabidopsis aspartic proteinase cleaved two other standard protein substrates at many sites, suggesting that this proteinase could have a role in the degradation of proteins in addition to processing propeptides in plants.

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Doi, Motomichi, Megumi Sato, and Yoshihiro Ohmiya. "In Vivo Simultaneous Analysis of Gene Expression by Dual-Color Luciferases in Caenorhabditis elegans." International Journal of Molecular Sciences 22, no. 1 (2020): 119. http://dx.doi.org/10.3390/ijms22010119.

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Both fluorescent and luminescent observation are widely used to examine real-time gene expression patterns in living organisms. Several fluuorescent and luminescent proteins with specific optical properties have been developed and applied for simultaneous, multi-color observation of more than two gene expression profiles. Compared to fluorescent proteins, however, the application of multi-color luminescent imaging in living organisms is still limited. In this study, we introduced two-color luciferases into the soil nematode C. elegans and performed simultaneous analysis of two gene expression profiles. Using a green-emitting luciferase Eluc (emerald luciferase) and red-emitting luciferase SLR (stable luciferase red), the expression patterns of two genes were simultaneously observed in single animals from embryonic to adult stages over its whole life span. In addition, dual gene activities were observed at the single embryo level, with the simultaneous observation of morphological changes. These are the first application of a two-color luciferase system into a whole animal and suggest that precise relationship of expression patterns of multiple genes of interest can be analyzed over the whole life of the animal, dependent on the changes in genetic and/or environmental conditions.

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Nakajima, Yoshihiro, and Yoshihiro Ohmiya. "Bioluminescence assays: multicolor luciferase assay, secreted luciferase assay and imaging luciferase assay." Expert Opinion on Drug Discovery 5, no. 9 (2010): 835–49. http://dx.doi.org/10.1517/17460441.2010.506213.

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Deeva, Anna A., Albert E. Lisitsa, Lev A. Sukovatyi, Tatiana N. Melnik, Valentina A. Kratasyuk, and Elena V. Nemtseva. "Structure-Function Relationships in Temperature Effects on Bacterial Luciferases: Nothing Is Perfect." International Journal of Molecular Sciences 23, no. 15 (2022): 8119. http://dx.doi.org/10.3390/ijms23158119.

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The evaluation of temperature effects on the structure and function of enzymes is necessary to understand the mechanisms underlying their adaptation to a constantly changing environment. In the current study, we investigated the influence of temperature variation on the activity, structural dynamics, thermal inactivation and denaturation of Photobacterium leiognathi and Vibrio harveyi luciferases belonging to different subfamilies, as well as the role of sucrose in maintaining the enzymes functioning and stability. We used the stopped-flow technique, differential scanning calorimetry and molecular dynamics to study the activity, inactivation rate, denaturation and structural features of the enzymes under various temperatures. It was found that P. leiognathi luciferase resembles the properties of cold-adapted enzymes with high activity in a narrow temperature range and slightly lower thermal stability than V. harveyi luciferase, which is less active, but more thermostable. Differences in activity at the studied temperatures can be associated with the peculiarities of the mobile loop conformational changes. The presence of sucrose does not provide an advantage in activity but increases the stability of the enzymes. Differential scanning calorimetry experiments showed that luciferases probably follow different denaturation schemes.

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Mazo-Vargas, Anyimilehidi, Heungwon Park, Mert Aydin, and Nicolas E. Buchler. "Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy." Molecular Biology of the Cell 25, no. 22 (2014): 3699–708. http://dx.doi.org/10.1091/mbc.e14-07-1187.

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Time-lapse fluorescence microscopy is an important tool for measuring in vivo gene dynamics in single cells. However, fluorescent proteins are limited by slow chromophore maturation times and the cellular autofluorescence or phototoxicity that arises from light excitation. An alternative is luciferase, an enzyme that emits photons and is active upon folding. The photon flux per luciferase is significantly lower than that for fluorescent proteins. Thus time-lapse luminescence microscopy has been successfully used to track gene dynamics only in larger organisms and for slower processes, for which more total photons can be collected in one exposure. Here we tested green, yellow, and red beetle luciferases and optimized substrate conditions for in vivo luminescence. By combining time-lapse luminescence microscopy with a microfluidic device, we tracked the dynamics of cell cycle genes in single yeast with subminute exposure times over many generations. Our method was faster and in cells with much smaller volumes than previous work. Fluorescence of an optimized reporter (Venus) lagged luminescence by 15–20 min, which is consistent with its known rate of chromophore maturation in yeast. Our work demonstrates that luciferases are better than fluorescent proteins at faithfully tracking the underlying gene expression.

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Viviani, Vadim R., T. L. Oehlmeyer, F. G. C. Arnoldi, and M. R. Brochetto-Braga. "A New Firefly Luciferase with Bimodal Spectrum: Identification of Structural Determinants of Spectral pH-Sensitivity in Firefly Luciferases¶." Photochemistry and Photobiology 81, no. 4 (2005): 843. http://dx.doi.org/10.1562/2004-12-09-ra-398r.1.

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Viviani, Vadim R., T. L. Oehlmeyer, F. G. C. Arnoldi, and M. R. Brochetto-Braga. "A New Firefly Luciferase with Bimodal Spectrum: Identification of Structural Determinants of Spectral pH-Sensitivity in Firefly Luciferases¶." Photochemistry and Photobiology 81, no. 4 (2007): 843–48. http://dx.doi.org/10.1111/j.1751-1097.2005.tb01452.x.

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Daniel, Catherine, Sabine Poiret, Véronique Dennin, Denise Boutillier, and Bruno Pot. "Bioluminescence Imaging Study of Spatial and Temporal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice." Applied and Environmental Microbiology 79, no. 4 (2012): 1086–94. http://dx.doi.org/10.1128/aem.03221-12.

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ABSTRACTLactic acid bacteria, especially lactobacilli, are common inhabitants of the gastrointestinal tract of mammals, for which they have received considerable attention due to their putative health-promoting properties. In this study, we describe the development and application of luciferase-expressingLactobacillus plantarumandLactococcus lactisstrains for noninvasivein vivomonitoring in the digestive tract of mice. We report for the first time the functionalin vitroexpression inLactobacillus plantarumNCIMB8826 and inLactococcus lactisMG1363 of the click beetle luciferase (CBluc), as well asGaussiaand bacterial luciferases, using a combination of vectors, promoters, and codon-optimized genes. We demonstrate that a CBluc construction is the best-performing luciferase system for the noninvasivein vivodetection of lactic acid bacteria after oral administration. The persistence and viability of both strains was studied by bioluminescence imaging in anesthetized mice and in mouse feces.In vivobioluminescence imaging confirmed that after a single or multiple oral administrations,L. lactishas shorter survival times in the mouse gastrointestinal tract thanL. plantarum, and it also revealed the precise gut compartments where both strains persisted. The application of luciferase-labeled bacteria has significant potential to allow thein vivoandex vivostudy of the interactions of lactic acid bacteria with their mammalian host.

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Karimi, Elham, Maryam Nikkhah, and Saman Hosseinkhani. "Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection." Biosensors 12, no. 11 (2022): 918. http://dx.doi.org/10.3390/bios12110918.

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A bioluminescence-based assay for ATP can measure cell viability. Higher ATP concentration indicates a higher number of living cells. Thus, it is necessary to design an ATP sensor that is low-cost and easy to use. Gold nanoparticles provide excellent biocompatibility for enzyme immobilization. We investigated the effect of luciferase proximity with citrate-coated gold, silver, and gold–silver core–shell nanoparticles, gold nanorods, and BSA–Au nanoclusters. The effect of metal nanoparticles on the activity of luciferases was recorded by the luminescence assay, which was 3–5 times higher than free enzyme. The results showed that the signal stability in presence of nanoparticles improved and was reliable up to 6 h for analytes measurements. It has been suggested that energy is mutually transferred from luciferase bioluminescence spectra to metal nanoparticle surface plasmons. In addition, we herein report the 27-base DNA aptamer for adenosine-5′-triphosphate (ATP) as a suitable probe for the ATP biosensor based on firefly luciferase activity and AuNPs. Due to ATP application in the firefly luciferase reaction, the increase in luciferase activity and improved detection limits may indicate more stability or accessibility of ATP in the presence of nanoparticles. The bioluminescence intensity increased with the ATP concentration up to 600 µM with a detection limit of 5 µM for ATP.

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Deane, Caitlin. "Luciferase matchmaker." Nature Chemical Biology 13, no. 4 (2017): 343. http://dx.doi.org/10.1038/nchembio.2352.

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Smale, S. T. "Luciferase Assay." Cold Spring Harbor Protocols 2010, no. 5 (2010): pdb.prot5421. http://dx.doi.org/10.1101/pdb.prot5421.

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Tsuji, Frederick I., E. Melvin Gindler, and David L. Davis. "LUCIFERASE-ANTILUCIFEFUSE*,†." Annals of the New York Academy of Sciences 103, no. 2 (2006): 715–23. http://dx.doi.org/10.1111/j.1749-6632.1963.tb53728.x.

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Bieleski, Lara, and Simon J. Talbot. "Kaposi's Sarcoma-Associated Herpesvirus vCyclin Open Reading Frame Contains an Internal Ribosome Entry Site." Journal of Virology 75, no. 4 (2001): 1864–69. http://dx.doi.org/10.1128/jvi.75.4.1864-1869.2001.

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ABSTRACT We have previously examined the transcription and splicing of open reading frames (ORFS) 71 (K13), 72, and 73 of Kaposi's sarcoma-associated herpesvirus (KSHV) in the primary effusion lymphoma cell line BCP-1 (latently infected with KSHV) (45). The three genes encoded by these ORFs (for vFLIP, vCyclin, and latency-associated nuclear antigen [LANA]) are transcribed from a common transcription start site in BCP-1 cells during both latency and the lytic cycles. The resulting transcript is spliced to yield a 5.32-kb message encoding LANA, vCyclin, and vFLIP and a 1.7-kb bicistronic message encoding vCyclin and vFLIP. To investigate whether the vFLIP protein could be expressed from this vCyclin/vFLIP message, we utilized a bicistronic luciferase reporter system. The genes for Renilla and firefly luciferases (which utilize different substrates) were cloned in tandem downstream from a T7 RNA polymerase promoter. Fragments of DNA immediately upstream from the initiating codon of vFLIP were cloned between the two luciferase genes. The relative expression of the two luciferases, one directed by the putative internal ribosome entry site (IRES) sequences and the other by cap-dependent ribosome scanning, was used to compare the activities of the different DNA fragments. A minimum fragment of 233 bp within the coding region of vCyclin was found to direct efficient expression of the downstream cistron (firefly luciferase). The activity of this IRES was orientation dependent and unaffected by methods used to inhibit cap-dependent translation. This is the first demonstration of an IRES element encoded by a DNA virus and may represent a novel mechanism through which KSHV controls protein expression.

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Silva Neto, A. J., V. Scorsato, F. G. C. Arnoldi, and V. R. Viviani. "Pyrearinus termitilluminans larval click beetle luciferase: active site properties, structure and function relationships and comparison with other beetle luciferases." Photochemical & Photobiological Sciences 8, no. 12 (2009): 1748. http://dx.doi.org/10.1039/b9pp00053d.

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Krasitskaya, Vasilisa V., Eugenia E. Bashmakova, and Ludmila A. Frank. "Coelenterazine-Dependent Luciferases as a Powerful Analytical Tool for Research and Biomedical Applications." International Journal of Molecular Sciences 21, no. 20 (2020): 7465. http://dx.doi.org/10.3390/ijms21207465.

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The functioning of bioluminescent systems in most of the known marine organisms is based on the oxidation reaction of the same substrate—coelenterazine (CTZ), catalyzed by luciferase. Despite the diversity in structures and the functioning mechanisms, these enzymes can be united into a common group called CTZ-dependent luciferases. Among these, there are two sharply different types of the system organization—Ca2+-regulated photoproteins and luciferases themselves that function in accordance with the classical enzyme–substrate kinetics. Along with deep and comprehensive fundamental research on these systems, approaches and methods of their practical use as highly sensitive reporters in analytics have been developed. The research aiming at the creation of artificial luciferases and synthetic CTZ analogues with new unique properties has led to the development of new experimental analytical methods based on them. The commercial availability of many ready-to-use assay systems based on CTZ-dependent luciferases is also important when choosing them by first-time-users. The development of analytical methods based on these bioluminescent systems is currently booming. The bioluminescent systems under consideration were successfully applied in various biological research areas, which confirms them to be a powerful analytical tool. In this review, we consider the main directions, results, and achievements in research involving these luciferases.

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Leach, Franklin R. "A View on the Active Site of Firefly Luciferase." Natural Product Communications 3, no. 9 (2008): 1934578X0800300. http://dx.doi.org/10.1177/1934578x0800300908.

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The bioluminescent enzyme firefly luciferase has been the subject of detailed biochemical studies since the late 1940s. These studies have culminated in the cloning and sequence determination of some 161 different c-DNAs and/or genes for firefly luciferase and the determination of 6 crystallographic structures with and without reactants/products/analogs bound. This paper reviews these studies, searches for which amino acid residues are involved in the enzyme's action using chemical modification experiments, and analyzes the structure/function relationships of the various conserved amino acid residues. There are 71/550 amino acid residues conserved in the 161 different firefly luciferases. In my laboratory K-206, K-529, and R-437 were chemically modified; ATP inhibited these modifications suggesting that they are involved in the reaction. An integrated picture of the active site is postulated.

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Zambito, Giorgia, Natasa Gaspar, Yanto Ridwan, et al. "Evaluating Brightness and Spectral Properties of Click Beetle and Firefly Luciferases Using Luciferin Analogues: Identification of Preferred Pairings of Luciferase and Substrate for In Vivo Bioluminescence Imaging." Molecular Imaging and Biology 22, no. 6 (2020): 1523–31. http://dx.doi.org/10.1007/s11307-020-01523-7.

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Abstract Purpose Currently, a variety of red and green beetle luciferase variants are available for bioluminescence imaging (BLI). In addition, new luciferin analogues providing longer wavelength luminescence have been developed that show promise for improved deep tissue imaging. However, a detailed assessment of these analogues (e.g., Akalumine-HCl, CycLuc1, and amino naphthyl luciferin (NH2-NpLH2)) combined with state of the art luciferases has not been performed. The aim of this study was to evaluate for the first time the in vivo brightness and spectral characteristics of firefly (Luc2), click beetle green (CBG99), click beetle red 2 (CBR2), and Akaluc luciferases when paired with different d-luciferin (d-LH2) analogues in vivo. Procedures Transduced human embryonic kidney (HEK 293T) cells expressing individual luciferases were analyzed both in vitro and in mice (via subcutaneous injection). Following introduction of the luciferins to cells or animals, the resulting bioluminescence signal and photon emission spectrum were acquired using a sensitive charge-coupled device (CCD) camera equipped with a series of band pass filters and spectral unmixing software. Results Our in vivo analysis resulted in four primary findings: (1) the best substrate for Luc2, CBG99, and CBR2 in terms of signal strength was d-luciferin; (2) the spectra for Luc2 and CBR2 were shifted to a longer wavelength when Akalumine-HCl was the substrate; (3) CBR2 gave the brightest signal with the near-infrared substrate, NH2-NpLH2; and (4) Akaluc was brighter when paired with either CycLuc1 or Akalumine-HCl when paired with d-LH2. Conclusion We believe that the experimental results described here should provide valuable guidance to end users for choosing the correct luciferin/luciferase pairs for a variety of BLI applications.

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Manukhov, I. V., and G. B. Zavilgelsky. "Biosensors for the Determination of Promoters and Chaperones Activity in Bacillus subtilis Cells." Biotekhnologiya 36, no. 6 (2020): 68–77. http://dx.doi.org/10.21519/0234-2758-2020-36-6-68-77.

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Plasmids to determine the activities of promoters in Bacillus subtilis cells have been constructed. Plasmids contain constitutive (PfbaA, PymdA_mut3, PymdA) and inducible (PxylA, PxylA-cre) promoters of different activities and genes under their control encoding reporter proteins with different thermostability and structure. The following main parameters were measured: maximal response, threshold concentration, minimal response time and the effect of catabolite repression for the inducible promoters. The activities of the constitutive promoters were compared. It was shown that the activity of the inducible PxylA promoter at a D(+)-xylose concentration of 1% was more than 100-fold higher and that of the PxylA-cre promoter was about 80-fold higher than the control level after 2 h of incubation. However, PxylA-cre is tightly closed and sensitive to glucose repression. The constructed lux sensors containing bacterial luciferases as reporter proteins were used to assess the influence of DnaKJE and trigger factor (TF) molecular chaperones on the level of synthesis of active enzymes. It was shown that the absence of both of the above chaperones in B. subtilis cells led to a significant decrease in the synthesis of the native thermolabile Photobacterium leiognathi luciferase, while the lack of TF increased the activity of the thermostable Photorhabdus luminescens luciferase by about 2 times as compared to wild type Bacillus subtilus 168. Erroneous translation under the streptomycin action and in the absence of both DnaKJE and TF chaperones significantly suppressed the synthesis of both luciferases. Bacillus subtilis, bacterial luciferase, biosensor, bioluminescence, promoter, chaperone, Trigger Factor, DnaKJ The work of I. Manukhov was supported by the Russian Science Foundation under the grant 20-16-00088. E. Gnuchikh's research on the design of lux-biosensors was partially funded by the Russian Foundation for Basic Research (project No. 20-34-70132).

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Leitão, João M. M., and Joaquim C. G. Esteves da Silva. "Firefly luciferase inhibition." Journal of Photochemistry and Photobiology B: Biology 101, no. 1 (2010): 1–8. http://dx.doi.org/10.1016/j.jphotobiol.2010.06.015.

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Hardy, Ralph W. F. "Let Out Luciferase." Nature Biotechnology 7, no. 9 (1989): 967. http://dx.doi.org/10.1038/nbt0989-967b.

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Bhaumik, S., X. Z. Lewis, and S. S. Gambhir. "Optical imaging of Renilla luciferase, synthetic Renilla luciferase, and firefly luciferase reporter gene expression in living mice." Journal of Biomedical Optics 9, no. 3 (2004): 578. http://dx.doi.org/10.1117/1.1647546.

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Omokoko, Tana A., Uli Luxemburger, Shaheer Bardissi, et al. "Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity." Journal of Immunology Research 2016 (2016): 1–13. http://dx.doi.org/10.1155/2016/9540975.

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Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughputin vitrocytotoxicity assays that efficiently analyze immune effector functions. The gold standard51Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferasein vitrotranscribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the51Cr release and further confirmed the assay’s ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay’s combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

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Viviani, Vadim R., and Etelvino J. H. Bechara. "BIOLUMINESCENCE OF BRAZILIAN FIREFLIES (COLEOPTERA: LAMPYRIDAE): SPECTRAL DISTRIBUTION and pH EFFECT ON LUCIFERASE-ELICITED COLORS. COMPARISON WITH ELATERID and PHENGODID LUCIFERASES." Photochemistry and Photobiology 62, no. 3 (1995): 490–95. http://dx.doi.org/10.1111/j.1751-1097.1995.tb02373.x.

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Inoue, Yusuke, Fugeng Sheng, Shigeru Kiryu, et al. "Gaussia Luciferase for Bioluminescence Tumor Monitoring in Comparison with Firefly Luciferase." Molecular Imaging 10, no. 5 (2011): 7290.2010.00057. http://dx.doi.org/10.2310/7290.2010.00057.

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Foucault, M. L., L. Thomas, S. Goussard, B. R. Branchini, and C. Grillot-Courvalin. "In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice." Applied and Environmental Microbiology 76, no. 1 (2009): 264–74. http://dx.doi.org/10.1128/aem.01686-09.

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ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

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Carvalho, M. C., A. Tomazini, D. T. Amaral, M. T. Murakami, and V. R. Viviani. "Luciferase isozymes from the Brazilian Aspisoma lineatum (Lampyridae) firefly: origin of efficient pH-sensitive lantern luciferases from fat body pH-insensitive ancestors." Photochemical & Photobiological Sciences 19, no. 12 (2020): 1750–64. http://dx.doi.org/10.1039/d0pp00272k.

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Chang, Dalu, Suihan Feng, Vladimir Girik, Howard Riezman, and Nicolas Winssinger. "Luciferase Controlled Protein Interactions." Journal of the American Chemical Society 143, no. 10 (2021): 3665–70. http://dx.doi.org/10.1021/jacs.0c11016.

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Bakhtiarova, Adel, Paul Taslimi, Stephen J. Elliman, et al. "Resveratrol inhibits firefly luciferase." Biochemical and Biophysical Research Communications 351, no. 2 (2006): 481–84. http://dx.doi.org/10.1016/j.bbrc.2006.10.057.

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Baldwin, Thomas O., Jon A. Christopher, Frank M. Raushel, et al. "Structure of bacterial luciferase." Current Opinion in Structural Biology 5, no. 6 (1995): 798–809. http://dx.doi.org/10.1016/0959-440x(95)80014-x.

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Chang, Dalu, Eric Lindberg, Suihan Feng, Simona Angerani, Howard Riezman, and Nicolas Winssinger. "Luciferase‐Induced Photouncaging: Bioluminolysis." Angewandte Chemie International Edition 58, no. 45 (2019): 16033–37. http://dx.doi.org/10.1002/anie.201907734.

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Chang, Dalu, Eric Lindberg, Suihan Feng, Simona Angerani, Howard Riezman, and Nicolas Winssinger. "Luciferase‐Induced Photouncaging: Bioluminolysis." Angewandte Chemie 131, no. 45 (2019): 16179–83. http://dx.doi.org/10.1002/ange.201907734.

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Abramenko, N. A., P. I. Vnukova, E. S. Golovina, et al. "Development and Validation of Approach for the Detection of Neutralizing Antibodies Against Insulin (Glargine) in Human Blood Plasma." Drug development & registration 8, no. 3 (2019): 70–78. http://dx.doi.org/10.33380/2305-2066-2019-8-3-70-78.

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Introduction. IImmunogenicity identification of therapeutic proteins, such as human insulin analogues, is one of the most relevant and significant area in medicine and pharmaceuticals. Determination the possibility of producing neutralizing antibodies to insulin reducing the therapeutic effect of the drug, is an important step to understand the pharmacological profile of the drug. Applying of cell-based methods one allows to determinate neutralizing antibodies to insulin.Aim. Development and validation methods for detection of neutralizing antibodies against insulin in human plasma.Materials and methods. The method is based on the use of the iLiteTM Insulin Assay Ready Cells [1], in the genome of which the firefly luciferase reporter gene is introduced under the control of an insulin-dependent promoter. As the insulin concentration increases, the firefly luciferase expression (Firefly) increases, allowing one to use this cell line to estimate the number of neutralizing antibodies against insulin. For normalization by the number of cells and considering the matrix effect of studied samples, the second reporter gene luciferase Renilla is used, which is expressed under the control of a constitutive promoter. The activity of both luciferases was measured using the DualGlo Luciferase Assay System (Promega) assay [2].Results and discussion. Optimal insulin concentration and plasma/serum dilution were determined to identify neutralizing antibodies to insulin. The long-term stability of neutralizing antibodies to insulin were shown in human plasma for more than 3 months. The developed method was applied in a comparative research of the safety and immunogenicity of insulin analogues (Glargine). Method for the determination of antibodies to insulin was.Conclusion. A method for determination of neutralizing antibodies to insulin in human K2EDTA plasma was developed and validated using iLiteTM Insulin Assay Ready Cells system; based on the binding of the insulin alpha chain to the high-affinity heterodimeric CD220 receptor.

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Journal articles on the topic 'Luciferasi'

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