For years, Ca2+-regulated photoproteins have been successfully used to detect calcium ions, ubiquitous eukaryotic intracellular messengers. Here we report the I144L obelin mutant with dramatically enhanced light output at low [Ca2+] which was unexpectedly found among the nineteen mutants with substitution of this conservative residue to all possible amino acids for all-round characterization. The mutant retains high specific activity, light signal kinetics, and spectral properties of wild-type obelin. Moreover, magnesium ions at physiological concentration of 1 mM do not significantly influence its bioluminescence activity. The OL I144L successfully demonstrated its potential as intracellular calcium reporter through assaying endogenous P2Y2 purinoreceptor activation in CHO cells by displaying a 2.4-fold higher sensitivity to calcium as compared to that of the wild-type obelin. The corresponding mutation was also introduced into the sequences of aequorin and clytin. Both mutants showed the enhanced bioluminescence activity at low [Ca2+] as compared to the corresponding wild-type photoproteins, but the increase of light signals in those was significantly lower than that of OL I144L mutant. Based on obelin structure, mutant properties, and the results obtained for other EF-hand Ca2+-binding proteins we speculate that the appearance of Leu instead of Ile affects the position of substrate within the inner cavity owing to a little different distances between Leu side chain and 2-hydroperoxycoelenterazine atoms which in turn may slightly shift the α-helices relative to each other changing the angles between them, thereby generating the 'pre-formed' EF-hand structures with a high affinity to calcium.

The functional screening of cDNA libraries (or functional cloning) enables isolation of cDNA genes encoding novel proteins with unknown amino acid sequences. This approach is the only way to identify a protein sequence in the event of shortage of biological material for obtaining pure target protein in amounts sufficient to determine its primary structure, since sensitive functional test for a target protein is only required to successfully perform functional cloning. Commonly, bioluminescent proteins from representatives belonging to different taxa significantly differ in sequences due to independent origin of bioluminescent systems during evolution. Nonetheless, these proteins are frequently similar in functions and can use even the same substrate of bioluminescence reaction, allowing the use of the same functional test for screening. The cDNA genes encoding unknown light-emitting proteins can be identified during functional screening with high sensitivity, which is provided by modern light recording equipment making possible the detection of a very small amount of a target protein. Here, we present the protocols for isolation of full-size cDNA genes for the novel bioluminescent protein family of light-sensitive Ca2+-regulated photoproteins in the absence of any sequence information by functional screening of plasmid cDNA expression library. The protocols describe all the steps from gathering animals to isolation of individual E. coli colonies carrying full-size cDNA genes using photoprotein berovin from ctenophore Beroe abyssicola as an illustrative example.

Light-sensitive Ca2+-regulated photoproteins of ctenophores are single-chain polypeptide proteins of 206-208 amino acids in length comprising three canonical EF-hand Ca2+-binding sites, each of 12 contiguous residues. These photoproteins are a stable complex of apoprotein and 2-hydroperoxy adduct of coelenterazine. Addition of calcium ions to photoprotein is only required to trigger bright bioluminescence. However, in contrast to the related Ca2+-regulated photoproteins of jellyfish their capacity to bioluminescence disappears on exposure to light over the entire absorption spectral range of ctenophore photoproteins. Here, we describe protocols for expression of gene encoding ctenophore photoprotein in Escherichia coli cells, obtaining of the recombinant apoprotein of high purity and its conversion into active photoprotein with synthetic coelenterazine as well as determination of its sensitivity to calcium ions using light-sensitive Ca2+-regulated photoprotein berovin from ctenophore Beroe abyssicola as an illustrative case.

Luciferases from copepods Metridia longa and Gaussia princeps are successfully used as bioluminescent reporters for in vivo and in vitro assays. Here, we report the minimal sequence of copepod luciferases required for bioluminescence activity that was revealed by gradual deletions of sequence encoding the smallest MLuc7 isoform of M. longa luciferase. The single catalytic domain is shown to reside within the G32-A149 MLuc7 sequence and to be formed by both non-identical repeats, including 10 conserved Cys residues. Because this part of MLuc7 displays high homology with those of other copepod luciferases, our suggestion is that the determined boundaries of the catalytic domain are the same for all known copepod luciferases. The involvement of the flexible C-terminus in the retention of the bioluminescent reaction product in the substrate-binding cavity was confirmed by structural modeling and kinetics study. We also demonstrate that the ML7-N10 mutant (15.4 kDa) with deletion of ten amino acid residues at the N-terminus can be successfully used as a miniature bioluminescent reporter in living cells. Application of a shortened reporter may surely reduce the metabolic load on the host cells and decrease steric and functional interference at its use as a part of hybrid proteins.

Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the “catalytic function” by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins.

The small coelenterazine-dependent luciferase from Metridia longa (MLuc), in view of its high activity, simplicity of bioluminescent (BL) reaction, and stability, has found successful analytical applications as a genetically encoded reporter for in vivo assessment of cellular processes. However, the study on the biochemical and BL properties and the development of in vitro analytical applications of MLuc are hampered by the difficulties of obtaining a sufficient amount of the highly active recombinant protein due to the presence of multiple (up to five) disulfide bonds per molecule. Here, we present a protocol to obtain the recombinant disulfide-rich MLuc using a cheap and simple Escherichia coli expression system without any affinity tags in its native form by refolding from inclusion bodies. The method includes (i) purification of MLuc inclusion bodies, solubilization of the aggregated form with full reduction of disulfide bonds, and refolding to the native state using a glutathione redox system in the presence of arginine and Cu2+ ions and (ii) chromatographic purification of MLuc and its functional assessment in terms of activity. We introduce the empirical, optimal conditions for oxidative refolding and subsequent purification of MLuc, with its basic properties taken into account. We believe that this protocol is adaptable for a large-scale harvest of other natively folded copepod luciferases as well as other disulfide-rich recombinant proteins from E. coli inclusion bodies.

Secreted copepod luciferases (CopLucs) represent highly homologous enzymes which catalyze the oxidation of a low molecular weight substrate, coelenterazine, with the emission of blue light (λmax = 485-488 nm), that is called bioluminescence (BL). The well-studied Gaussia (GLuc) and Metridia (MLuc) luciferases originally cloned from the marine copepods Gaussia princeps and Metridia longa belong to the group of the smallest natural luciferases. Their minimal molecular weight, high luminescent activity, cofactor-independent BL, and the ability to be secreted due to the own signal peptide open up the horizons for genetic engineering of CopLuc-based sensitive biosensors for in vivo imaging and in vitro analytical applications. The "standard" soluble bacterial expression of the recombinant CopLucs and luciferase-based hybrid proteins is hampered by the presence of high amounts of intramolecular disulfide bonds (up to 5 per molecule). Here, we describe the universal protocol for highly effective secreted expression of disulfide-rich CopLucs using their own signal peptide in insect cells and their purification from serum-free culture medium. The suggested protocol allows obtaining high-purity CopLucs folded in their native form with the yield of up to 5 mg per liter.

MicroRNAs epigenetically regulate physiological and pathological processes. Previously, we found that miR-204-5p is expressed at low levels in melanoma cells, and an increase in its level leads to a change in proliferation, migration, and invasion of these cancer cells. Now, using bioinformatics analysis, it has been shown that the target of miR-204-5p is FOXC1 transcription factor, which is implicated in carcinogenesis. Using the luciferase reporter assay, it was found that miR-204-5p suppresses expression of the FOXC1 gene by binding to its 3' non-coding region. Transfection of small interfering RNA (siRNA) targeting FOXC1 into melanoma cells caused a decrease in miR-204-5p levels, which is consistent with the generally accepted concept of feedback regulation of miRNA expression by target genes. According to the results of the MTT test and fluorescence microscopy, the proliferation level of melanoma cells under the influence of siRNA to FOXC1 decreased 72 h after transfection. Changes in the ratio of cells by cell cycle phase were analyzed using flow cytometry. Regulatory relationships between FOXC1 and miR-204-5p, and an inhibitory effect of FOXC1 knockdown on melanoma cell proliferation were revealed. Based on the results, it can be assumed that miR-204-5p regulates proliferation of melanoma cells by affecting FOXC1 expression.

MicroRNAs epigenetically regulate physiological and pathological processes. Previously, we found that miR-204-5p is expressed at low levels in melanoma cells, and an increase in its level leads to a change in proliferation, migration, and invasion of these cancer cells. Now, using bioinformatics analysis, it has been shown that the target of miR-204-5p is FOXC1 transcription factor, which is implicated in carcinogenesis. Using the luciferase reporter assay, it was found that miR-204-5p suppresses expression of the FOXC1 gene by binding to its 3' non-coding region. Transfection of small interfering RNA (siRNA) targeting FOXC1 into melanoma cells caused a decrease in miR-204-5p levels, which is consistent with the generally accepted concept of feedback regulation of miRNA expression by target genes. According to the results of the MTT test and fluorescence microscopy, the proliferation level of melanoma cells under the influence of siRNA to FOXC1 decreased 72 h after transfection. Changes in the ratio of cells by cell cycle phase were analyzed using flow cytometry. Regulatory relationships between FOXC1 and miR-204-5p, and an inhibitory effect of FOXC1 knockdown on melanoma cell proliferation were revealed. Based on the results, it can be assumed that miR-204-5p regulates proliferation of melanoma cells by affecting FOXC1 expression.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepodMetridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (K-D= 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (K-D= 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform ofM. longaluciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

Bioluminescent proteins are widely used as reporter molecules in various in vitro and in vivo assays. The smallest isoform of Metridia luciferase (MLuc7) is a highly active, naturally secreted enzyme which, along with other luciferase isoforms, is responsible for the bright bioluminescence of marine copepod Metridia longa. In this study, we report the construction of two variants of a hybrid protein consisting of MLuc7 and 14D5a single-chain antibody to the surface glycoprotein E of tick-borne encephalitis virus as a model fusion partner. We demonstrate that, whereas fusion of a single-chain antibody to either N- or C-terminus of MLuc7 does not affect its bioluminescence properties, the binding site on the single-chain antibody influences its binding capacity. The affinity of 14D5a-MLuc7 hybrid protein (KD = 36.2 nM) where the C-terminus of the single-chain antibody was fused to the N-terminus of MLuc7, appeared to be 2.5-fold higher than that of the reverse, MLuc7-14D5a (KD = 87.6 nM). The detection limit of 14D5a-MLuc7 hybrid protein was estimated to be 45 pg of the recombinant glycoprotein E. Although the smallest isoform of M. longa luciferase was tested as a fusion partner only with a single-chain antibody, it is reasonable to suppose that MLuc7 can also be successfully used as a partner for genetic fusion with other proteins.

Copepod luciferases-a family of small secretory proteins of 18.4-24.3 kDa, including a signal peptide-are responsible for bright secreted bioluminescence of some marine copepods. The copepod luciferases use coelenterazine as a substrate to produce blue light in a simple oxidation reaction without any additional cofactors. They do not share sequence or structural similarity with other identified bioluminescent proteins including coelenterazine-dependent Renilla and Oplophorus luciferases. The small size, strong luminescence activity and high stability, including thermostability, make secreted copepod luciferases very attractive candidates as reporter proteins which are particularly useful for nondisruptive reporter assays and for high-throughput format. The most known and extensively investigated representatives of this family are the first cloned GpLuc and MLuc luciferases from copepods Gaussia princeps and Metridia longa, respectively. Immediately after cloning, these homologous luciferases were successfully applied as bioluminescent reporters in vivo and in vitro, and since then, the scope of their applications continues to grow. This review is an attempt to systemize and critically evaluate the data scattered through numerous articles regarding the main structural features of copepod luciferases, their luminescent and physicochemical properties. We also review the main trends of their application as bioluminescent reporters in cell and molecular biology.

The secreted luciferases responsible for light emission of marine copepods have gained popularity for being used in noninvasive imaging of intracellular events. The secreted luciferase of copepod Gaussia princeps is a one subunit protein catalyzing coelenterazine oxidation to emit blue light. It consists of the N-terminal variable part that bears a signal peptide for secretion and the C-terminal catalytic domain containing ten highly conserved Cys residues supposing the existence of up to five S-S bonds. Despite wide application of Gaussia luciferase in biomedical research, its biochemical properties are still insufficiently studied due to the general problem of obtaining the proper folded Cys-rich proteins in bacterial cells. Here we report the properties of the proper folded Gaussia luciferase produced in insect cells using baculovirus expression system. This high purity luciferase reveals the highest activity at 15-20 degrees C but retains only similar to 20% activity at 37 degrees C that may hamper its application for in vivo assays. The maximum of bioluminescent activity of GpLuc is found at NaCl concentrations in the range of 1.0-1.5 M and, furthermore, a high NaCl concentration enhances luciferase stability to thermal denaturation, i.e. Gaussia luciferase displays the features characteristic of halophilic enzymes. The studies on bioluminescence kinetics at different coelenterazine concentrations obviously show a positive cooperativity of Gaussia luciferase with coelenterazine (Hill coefficient -1.8 +/- 0.2; K-0.5-2.14 +/- 0.17 mu M). We suggest this effect to be rather due to the so-called kinetic cooperativity conditioned by conformational changes in response to substrate binding than to the presence of two catalytic sites.

Нейронная сеть представляет собой математическую модель и ее воплощение в виде программной либо программно-аппаратной реализации, которая выполнена на основе моделирования деятельности биологических нейронных сетей, которыми являются сети нейронов в биологическом организме. Научный интерес к данной структуре возник потому, что исследование ее модели позволяет получать информацию о некоторой системе. То есть подобная модель может иметь практическую реализацию в ряде отраслей современной науки и техники. В статье рассматриваются вопросы, затрагивающие тему применения нейронных сетей для построения систем идентификации изображений, имеющих широкое применение в системах обеспечения безопасности. Подробно исследованы вопросы, связанные с темой алгоритма распознавания изображения и его применения. Кратко приведена информация о методике обучения нейронных сетей.

Secreted reporter proteins provide monitoring of intracellular events in real time without cell destruction. To create human melanoma cell lines that enables noninvasive bioluminescent monitoring of metabolic activity, a comparison of the efficiency of isoforms and mutant variants of luciferase from the Metridia longa as secreted reporter proteins in the cells of human melanoma lines Mel IL was conducted. The MLM3 deletion mutant had the highest activity in the medium of two studied isoforms and two deletion mutants of secreted M. longa luciferase during the Mel IL melanoma cell transfection. It was established that optimization of the gene structure of the selected MLM3 variant for expression in human cells increases the level of bioluminescent activity in the Mel IL cells by almost an order of magnitude. A stable Mel IL melanoma cell line with constitutive expression of the humanized hMLM3 reporter gene was obtained and characterized. The linear range of identification of living cells by the hMLM3 reporter activity was more than three orders of magnitude with a sensitivity of detection of 10 cells.

The bioluminescence of a marine copepod Metridia longa is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (λmax = 480 nm). To date, four different isoforms of the luciferase differing in size, sequences, and properties have been cloned by functional screening. All of them contain ten conserved Cys residues that suggests up to five SS intramolecular bonds per luciferase molecule. Whereas the use of copepod luciferases as bioluminescent reporters in biomedical research in vivo is growing from year to year, their application for in vitro assays is still limited by the difficulty in obtaining significant amounts of luciferase. The most cost-effective host for producing recombinant proteins is Escherichia coli. However, prokaryotic and eukaryotic cells maintain the reductive environment in cytoplasm that hinders the disulfide bond formation and consequently the proper folding of luciferase. Here we report the expression of the MLuc7 isoform of M. longa luciferase in E. coli cells and the efficient procedure for refolding from inclusion bodies yielding a high-active monomeric protein. Furthermore, in a set of identical experiments we demonstrate that bioluminescent and structural features of MLuc7 produced in bacterial cells are identical to those of MLuc7 isoform produced from culture medium of insect cells. Although the yield of high-purity protein is only 6 mg/L, the application of E. coli cells to produce the luciferase is simpler and more cost-effective than the use of insect cells. We expect that the suggested technology of Metridia luciferase production allows obtaining of sufficient amounts of protein both for the development of novel in vitro analytical assays with the use of MLuc7 as a label and for structural studies.