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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Bashmakova E. E., Krasitskaya V. V., Kudryavtsev A. N., Grigorenko V. G., Frank L. A.
Заглавие : Hybrid Minimal Core Streptavidin–Obelin as a Versatile Reporter for Bioluminescence-based Bioassay
Место публикации : Photochem. Photobiol.: Blackwell Publishing Inc., 2017. - Vol. 93, Is. 2. - С. 548-552. - ISSN 00318655 (ISSN) , DOI 10.1111/php.12648
Аннотация: Ca2+-regulated photoprotein obelin was genetically fused with a minimum-sized core streptavidin. Hybrid protein (SAV–OL) was produced by bacterial expression and applied as a specific bioluminescent probe in diverse solid-phase assays. The obtained results clearly demonstrate specific activity of each domain indicating its proper folding with favorable space orientation. SAV–OL has been shown to be a much more sensitive label than the chemical conjugate of a full-length streptavidin with obelin. © 2016 The American Society of Photobiology
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Larionova, Marina D., Markova, Svetlana V., Vysotski, Eugene S.
Заглавие : The novel extremely psychrophilic luciferase from Metridia longa: Properties of a high-purity protein produced in insect cells
Колич.характеристики :7 с
Коллективы : Bayer AG (Germany); Russian Academy of Sciences [01201351504]; RFBR; Government of Krasnoyarsk Territory [16-44-242099]
Место публикации : Biochem. Biophys. Res. Commun.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017. - Vol. 483, Is. 1. - С. 772-778. - ISSN 0006-291X, DOI 10.1016/j.bbrc.2016.12.067. - ISSN 1090-2104(eISSN)
Примечания : Cited References:24. - The cloning of cDNAs encoding MLuc2 isoforms of M. longa was supported by Bayer AG (Germany) and the state budget allocated to the fundamental research at the Russian Academy of Sciences (project No. 01201351504); all other studies were funded by RFBR and Government of Krasnoyarsk Territory according to the research project No. 16-44-242099.
Предметные рубрики: CDNA CLONING
EXPRESSION
ENZYME
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--bioluminescent reporter--psychrophilic--enzyme--molecular adaptation
Аннотация: The bright bioluminescence of copepod Metridia longa is conditioned by a small secreted coelenterazinedependent luciferase (MLuc). To date, three isoforms of MLuc differing in length, sequences, and some properties were cloned and successfully applied as high sensitive bioluminescent reporters. In this work, we report cloning of a novel group of genes from M. longa encoding extremely psychrophilic isoforms of MLuc (MLuc2-type). The novel isoforms share only similar to 54-64% of protein sequence identity with the previously cloned isoforms and, consequently, are the product of a separate group of paralogous genes. The MLuc2 isoform with consensus sequence was produced as a natively folded protein using baculovirus/ insect cell expression system, purified, and characterized. The MLuc2 displays a very high bioluminescent activity and high thermostability similar to those of the previously characterized M. longa luciferase isoform MLuc7. However, in contrast to MLuc7 revealing the highest activity at 12-17 degrees C and 0.5 M NaCl, the bioluminescence optima of MLuc2 isoforms are at similar to 5 degrees C and 1 M NaCl. The MLuc(2) adaptation to cold is also accompanied by decrease of melting temperature and affinity to substrate suggesting a more conformational flexibility of a protein structure. The luciferase isoforms with different temperature optima may provide adaptability of the M. longa bioluminescence to the changes of water temperature during diurnal vertical migrations. (C) 2016 Elsevier Inc. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Krasitskaya, Vasilisa V., Chaukina, Valentina V., Abroskina, Maria V., Vorobyeva, Maria A., Ilminskaya, Aleksandra A., Kabilov, Marsel R., Prokopenko, Semyon V., Nevinsky, Georgy A., Venyaminova, Alya G., Frank, Ludmila A.
Заглавие : Bioluminescent aptamer-based sandwich-type assay of anti-myelin basic protein autoantibodies associated with multiple sclerosis
Колич.характеристики :7 с
Коллективы : Russian Foundation for Basic Research (RFBR), Russia [17-315-50027]; Russian State [AAAA-A17-117013050026-4, AAAA-A17-117020210021-7]
Место публикации : Anal. Chim. Acta: ELSEVIER SCIENCE BV, 2019. - Vol. 1064. - С. 112-118. - ISSN 0003-2670, DOI 10.1016/j.aca.2019.03.015. - ISSN 1873-4324(eISSN)
Примечания : Cited References:29. - This work was supported by the Russian Foundation for Basic Research (RFBR), Russia, under the grant No 17-315-50027; Russian State funded budget projects No. AAAA-A17-117013050026-4 and AAAA-A17-117020210021-7.
Предметные рубрики: ANTIBODIES
BIOMARKERS
RNA
Ключевые слова (''Своб.индексиров.''): bioluminescent microassay--rna aptamers--autoantibodies to myelin basic--protein--multiple sclerosis
Аннотация: Bioluminescent solid-phase sandwich-type microassay was developed to detect multiple sclerosis (MS)-associated autoantibodies in human sera. The assay is based on two different 2'-F-Py RNA aptamers against the target autoantibodies as biospecific elements, and Ca2+-regulated photoprotein obelin as a reporter. The paper describes elaboration of the assay and its application to 91 serum samples from patients with clinically definite MS and 86 ones from individuals healthy in terms of MS. Based on the receiver-operator curve (ROC) analysis, the chosen threshold value as clinical decision limit offers sensitivity of 63.7% and specificity of 94.2%. The area under the ROC curve (AUC) value of 0.87 shows a good difference between the groups under investigation. The likelihood ratio of 10.97 proves the diagnostic value of the assay and its potential as one of the laboratory MS-tests. (C) 2019 Elsevier B.V. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova, Svetlana V., Larionova, Marina D., Vysotski, Eugene S.
Заглавие : Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications.
Место публикации : Photochemistry and photobiology. - 2018. - ISSN 1751-1097, DOI 10.1111/php.13077
Аннотация: 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. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Frank, Ludmila A.
Заглавие : Creation of Artificial Luciferases to Expand their Analytical Potential
Колич.характеристики :11 с
Коллективы : RFBR [14-08-00902/14]; [VI 57.1.1]
Место публикации : Comb. Chem. High Throughput Screen: BENTHAM SCIENCE PUBL LTD, 2015. - Vol. 18, Is. 10. - С. 919-929. - ISSN 1386-2073, DOI 10.2174/1386207318666150917100011. - ISSN 1875-5402(eISSN)
Примечания : Cited References:79. - The work was supported by: the RFBR grant No. 14-08-00902/14; the State budget allocated to the fundamental research at the Russian Academy of Sciences (project No. VI 57.1.1).
Предметные рубрики: BIOLUMINESCENT REPORTER APPLICATIONS
COELENTERAZINE-BINDING PROTEIN
Ключевые слова (''Своб.индексиров.''): luciferase--luciferin--photoprotein--bioluminescence--mutagenesis--luciferase-based assay--bioimaging--reporter assay
Аннотация: Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescence-based analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Burakova, Ludmila P., Kudryavtsev, Alexander N., Stepanyuk, Galina A., Baykov, Ivan K., Morozova, Vera V., Tikunova, Nina V., Dubova, Maria A., Lyapustin, Victor N., Yakimenko, Valeri V., Frank, Ludmila A.
Заглавие : Bioluminescent detection probe for tick-borne encephalitis virus immunoassay
Колич.характеристики :7 с
Коллективы : Russian Academy of Sciences, Siberian Branch [139], Russian Academy of Sciences [VI 57.1.1]
Место публикации : Anal. Bioanal. Chem.: SPRINGER HEIDELBERG, 2015. - Vol. 407, Is. 18. - С. 5417-5423. - ISSN 1618-2642, DOI 10.1007/s00216-015-8710-6. - ISSN 1618-2650(eISSN)
Примечания : Cited References:19. - The work was supported by the Russian Academy of Sciences, Siberian Branch, within the framework of the Interdisciplinary Integration Project No. 139 and the State budget allocated to the fundamental research at the Russian Academy of Sciences (project No. VI 57.1.1).
Предметные рубрики: COELENTERAZINE-BINDING PROTEIN
ENZYME-IMMUNOASSAY
RENILLA-MUELLERI
Ключевые слова (''Своб.индексиров.''): tick-borne encephalitis virus--single-chain antibody--luciferase--immunoassay
Аннотация: To facilitate the detection of the tick-borne encephalitis virus (TBEV), the causative agent of one of the most severe human neuroinfections, we have developed an immunoassay based on bioluminescent hybrid protein 14D5a-Rm7 as a detection probe. The protein containing Renilla luciferase as a reporter and a single-chain variable fragment (scFv) of murine immunoglobulin to TBEV as a recognition element was constructed, produced by bacterial expression, purified, and tested. Both domains were shown to reveal their specific biological properties-affinity to the target antigen and bioluminescent activity. Hybrid protein was applied as a label for solid-phase immunoassay of the antigens, associated with the tick-borne encephalitis virus (native glycoprotein E or extracts of the infected strain of lab ticks). The assay demonstrates high sensitivity (0.056 ng of glycoprotein E; 10(4)-10(5) virus particles or 0.1 pg virions) and simplicity and is competitive with conventional methods for detection of TBEV.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Deeva, Anna A., Temlyakova, Evgenia A., Sorokin, Anatoly A., Nemtseva, Elena V., Kratasyuk, Valentina A.
Заглавие : Structural distinctions of fast and slow bacterial luciferases revealed by phylogenetic analysis
Колич.характеристики :5 с
Коллективы : RFBR [16-34-00746 mol_a]; Ministry of Education and Science of the Russian Federation [1762]; state budget allocated to the fundamental research at Russian Academy of Sciences [01201351504]
Место публикации : Bioinformatics: OXFORD UNIV PRESS, 2016. - Vol. 32, Is. 20. - С. 3053-3057. - ISSN 1367-4803, DOI 10.1093/bioinformatics/btw386. - ISSN 1460-2059(eISSN)
Примечания : Cited References:31. - The reported study was partially funded by RFBR according to the research project No. 16-34-00746 mol_a; by the Ministry of Education and Science of the Russian Federation [project No 1762] and by the state budget allocated to the fundamental research at the Russian Academy of Sciences [project No 01201351504].
Аннотация: Motivation: Bacterial luciferases are heterodimeric enzymes that catalyze a chemical reaction, so called bioluminescence, which causes light emission in bacteria. Bioluminescence is vastly used as a reporter system in research tools and commercial developments. However, the details of the mechanisms that stabilize and transform the reaction intermediates as well as differences in the enzymatic kinetics amongst different bacterial luciferases remain to be elucidated. Results: Amino acid sequences alignments for 21 bacterial luciferases (both alpha- and beta-subunits) were analyzed. For alpha-subunit, containing the enzyme active center, 48 polymorphic amino acid positions were identified. According to them, the sequences fell into two distinct groups known as slow and fast based on the decay rate of the bioluminescence reaction. The differences in the enzyme active site induced by structural polymorphism are analyzed.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Frank L. A.
Заглавие : Creation of artificial luciferases to expand their analytical potential
Место публикации : Comb. Chem. High Throughput Screen. - 2015. - Vol. 18, Is. 10. - С. 919-929. - ISSN 13862073 (ISSN)
Ключевые слова (''Своб.индексиров.''): bioimaging--bioluminescence--luciferase--luciferase-based assay--luciferin--mutagenesis--photoprotein--reporter assay
Аннотация: Bioluminescent proteins have been intensively used as high sensitive reporters in all kinds of binding assays (immuno-, nucleic acid hybridization assays, etc.) and in bioimaging. But natural luciferases do not always meet the requirements set for them as the assay reporters: thermostabitity, definite bioluminescence spectral and kinetics characteristics, stability to chemical modifications, etc. Luciferases with different appropriate characteristics as well as various luciferin derivatives were obtained using mutagenesis and chemical synthesis. Thanks to rigorous efforts of many researchers bioluminescencebased analytical techniques offer a great potential for solving analytical tasks in the field of biotechnology, biomedicine, pharmacology, etc. © 2015 Bentham Science Publishers.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Dubovtseva I. Y., Aksenenko M. B., Nikolaeva E. D., Averchuk A. S., Moshev A. V., Savchenko A. A., Markova S. V., Ruksha T. G.
Заглавие : FOXC1-Mediated Effects of miR-204-5p on Melanoma Cell Proliferation
Место публикации : Mol Biol (Mosk): NLM (Medline), 2021. - Vol. 55, Is. 4. - С. 667-675. - ISSN 00268984 (ISSN), DOI 10.31857/S0026898421030058
Аннотация: 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.
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10.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Dubovtseva, I. Yu, Aksenenko M. B., Nikolaeva E. D., Averchuk A. S., Moshev, A., V, Savchenko A. A., Markova, S., V, Ruksha T. G.
Заглавие : FOXC1-Mediated Effects of miR-204-5p on Melanoma Cell Proliferation
Колич.характеристики :8 с
Место публикации : Mol. Biol.: PLEIADES PUBLISHING INC, 2021. - Vol. 55, Is. 4. - С. 610-617. - ISSN 0026-8933, DOI 10.1134/S0026893321020199. - ISSN 1608-3245(eISSN)
Примечания : Cited References:24
Предметные рубрики: FOXC1
Аннотация: 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.
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11.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Nemtseva, Elena, V, Gulnov, Dmitry, V, Gerasimova, Marina A., Sukovatyi, Lev A., Burakova, Ludmila P., Karuzina, Natalya E., Melnik, Bogdan S., Kratasyuk, Valentina A.
Заглавие : Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
Колич.характеристики :17 с
Коллективы : Ministry of Science and Higher Education of the Russian Federation [FSRZ-2020-0006]; RFBRRussian Foundation for Basic Research (RFBR) [20-34-90118]; Krasnoyarsk Regional Fund of Science [20-44-243002, 20-44-240006]; RFBRRussian Foundation for Basic Research (RFBR)
Место публикации : Int. J. Mol. Sci.: MDPI, 2021. - Vol. 22, Is. 19. - Ст.10449. - ISSN 1422-0067(eISSN), DOI 10.3390/ijms221910449
Примечания : Cited References:45. - The research was partially funded by the Ministry of Science and Higher Education of the Russian Federation (projects No. FSRZ-2020-0006); by the RFBR and Krasnoyarsk Territory and Krasnoyarsk Regional Fund of Science (projects No. 20-44-243002 and 20-44-240006); and by the RFBR (project No. 20-34-90118).
Предметные рубрики: TRYPTOPHAN FLUORESCENCE
CRYSTAL-STRUCTURE
SUBUNIT
BIOLUMINESCENCE
Аннотация: 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 alpha-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./p
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12.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Nemtseva E. V., Gulnov D. V., Gerasimova M. A., Sukovatyi L. A., Burakova L. P., Karuzina N. E., Melnik B. S., Kratasyuk V. A.
Заглавие : Bacterial luciferases from vibrio harveyi and photobacterium leiognathi demonstrate different conformational stability as detected by time-resolved fluorescence spectroscopy
Место публикации : Int. J. Mol. Sci.: MDPI, 2021. - Vol. 22, Is. 19. - Ст.10449. - ISSN 16616596 (ISSN), DOI 10.3390/ijms221910449
Аннотация: 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 ?-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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.
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13.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Larionova M. D., Markova S. V., Tikunova N. V., Vysotski E. S.
Заглавие : The smallest isoform of Metridia longa luciferase as a fusion partner for hybrid proteins
Место публикации : Int. J. Mol. Sci.: MDPI AG, 2020. - Vol. 21, Is. 14. - Ст.4971. - С. 1-16. - ISSN 16616596 (ISSN), DOI 10.3390/ijms21144971
Аннотация: 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. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
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14.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Krasitskaya, Vasilisa V., Goncharova, Natalia S., Biriukov, Vladislav V., Bashmakova, Eugenia E., Kabilov, Marsel R., Baykov, Ivan K., Sokolov, Aleksey E., Frank, Ludmila A.
Заглавие : The Ca2+-Regulated Photoprotein Obelin as a Tool for SELEX Monitoring and DNA Aptamer Affinity Evaluation
Колич.характеристики :6 с
Коллективы : Russian Foundation for Basic Research (RFBR)Russian Foundation for Basic Research (RFBR) [18-38-00531]
Место публикации : Photochem. Photobiol.: WILEY, 2020. - Article in press. - ISSN 0031-8655, DOI 10.1111/php.13274. - ISSN 1751-1097(eISSN)
Примечания : Cited References:25. - This work has been supported by the Russian Foundation for Basic Research (RFBR) under the grant no 18-38-00531.
Предметные рубрики: CARDIAC TROPONIN-I
BIOLUMINESCENCE
IMMUNOASSAY
APTASENSOR
DIAGNOSIS
Аннотация: Bioluminescent solid-phase analysis was proposed to monitor the selection process and to determine binding characteristics of the aptamer-target complexes during design and development of the specific aptamers. The assay involves Ca2+-regulated photoprotein obelin as a simple, sensitive and fast reporter. Applicability and the prospects of the approach were exemplified by identification of DNA aptamers to cardiac troponin I, a highly specific early biomarker for acute myocardial infarction. Two structurally different aptamers specific to various epitopes of troponin I were obtained and then tested in a model bioluminescent assay.
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15.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Davydova, Anna, Krasitskaya, Vasilisa, Vorobjev, Pavel, Timoshenko, Valentina, Tupikin, Alexey, Kabilov, Marsel, Frank, Ludmila, Venyaminova, Alya, Vorobyeva, Mariya
Заглавие : Reporter-recruiting bifunctional aptasensor for bioluminescent analytical assays
Колич.характеристики :7 с
Коллективы : Russian Science FoundationRussian Science Foundation (RSF) [16-14-10296]; Russian State funded budget projects [AAAA-A17-117020210021-7, AAAA-A19-119031890015-0]
Место публикации : RSC Adv.: ROYAL SOC CHEMISTRY, 2020. - Vol. 10, Is. 54. - С. 32393-32399. - ISSN 2046-2069(eISSN), DOI 10.1039/d0ra05117a
Примечания : Cited References:33. - The work was supported by the Russian Science Foundation (grant #16-14-10296), Russian State funded budget projects #AAAA-A17-117020210021-7 to ICBFM SB RAS and #AAAA-A19-119031890015-0 to IBP SB RAS.
Предметные рубрики: DNA APTAMER
RNA APTAMER
OBELIN
PURIFICATION
EXPRESSION
SEQUENCES
Аннотация: We report a novel bioluminescent aptasensor, which consists of 2 '-F-RNA aptamer modules joined into a bi-specific aptamer construct. One aptamer module binds the analyte, then after structural rearrangement the second module recruits non-covalently Ca2+-dependent photoprotein obelin from the solution, thus providing a bioluminescent signal. This concept allows using free protein as a reporter, which brings such advantages as no need for aptamer-protein conjugation, a possibility of thermal re-folding of aptamer component with no harm to a protein, and simpler detection protocol. We developed the new 2 '-F-RNA aptamer for obelin, and proposed the strategy for engineering structure-switching bi-modular aptamer constructs which bind the analyte and the obelin in a sequential manner. With the use of hemoglobin as a model analyte, we showed the feasibility of utilizing the aptasensor in a fast and straightforward bioluminescent microplate assay. With a proper design of a secondary structure, this strategy of aptasensor engineering might be further extended to bi-specific aptamer-based bioluminescent sensors for other analytes of interest.
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16.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Larionova, Marina D., Markova, Svetlana, V, Tikunova, Nina, V, Vysotski, Eugene S.
Заглавие : The Smallest Isoform ofMetridia longaLuciferase as a Fusion Partner for Hybrid Proteins
Колич.характеристики :16 с
Коллективы : Russian Foundation for Basic ResearchRussian Foundation for Basic Research (RFBR) [18-44-242001]; Krasnoyarsk Regional Fund of Science; ICBFM SB RAS [AAAA-A17-117020210027-9]; Government of Krasnoyarsk Territory
Место публикации : Int. J. Mol. Sci.: MDPI, 2020. - Vol. 21, Is. 14. - Ст.4971. - ISSN 1422-0067(eISSN), DOI 10.3390/ijms21144971
Примечания : Cited References:49. - The reported study was funded by the Russian Foundation for Basic Research (No. 18-44-242001), Government of Krasnoyarsk Territory, Krasnoyarsk Regional Fund of Science (S.V.M, M.D.L., and E.S.V.) and by the Russian State funded budget project of ICBFM SB RAS No. AAAA-A17-117020210027-9 (N.V.T.).
Предметные рубрики: COELENTERAZINE-BINDING PROTEIN
BIOLUMINESCENT REPORTER
GAUSSIA
Аннотация: 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.
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17.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova, Svetlana V., Larionova, Marina D., Burakova, Ludmila P., Vysotski, Eugene S.
Заглавие : The smallest natural high-active luciferase: Cloning and characterization of novel 16.5-kDa luciferase from copepod Metridia longa
Колич.характеристики :6 с
Коллективы : Bayer AG (Germany); Russian Science Foundation [14-14-01119]
Место публикации : Biochem. Biophys. Res. Commun.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2015. - Vol. 457, Is. 1. - С. 77-82. - ISSN 0006-291X, DOI 10.1016/j.bbrc.2014.12.082. - ISSN 1090-2104(eISSN)
Примечания : Cited References:20. - The cloning of cDNA encoding MLuc7 luciferase of M. longa was supported by Bayer AG (Germany); all other studies - by the grant 14-14-01119 of the Russian Science Foundation. We declare that authors have no conflict of interest.
Предметные рубрики: CDNA CLONING
SECRETED LUCIFERASE
ESCHERICHIA-COLI
EXPRESSION
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--copepod luciferase--mammalian--expression--real-time imaging
Аннотация: Coelenterazine-dependent copepod luciferases containing natural signal peptide for secretion are a very convenient analytical tool as they enable monitoring of intracellular events with high sensitivity, without destroying cells or tissues. This property is well suited for application in biomedical research and development of cell-based assays for high throughput screening. We report the cloning of cDNA gene encoding a novel secreted non-allelic 16.5-kDa isoform (MLuc7) of Metridia longa luciferase, which, in fact, is the smallest natural luciferase of known for today. Despite the small size, isoform contains 10 conservative Cys residues suggesting the presence of up to 5 S-S bonds. This hampers the efficient production of functionally active recombinant luciferase in bacterial expression systems. With the use of the baculovirus expression system, we produced substantial amounts of the proper folded MLuc7 luciferase with a yield of similar to 3 mg/L of a high purity protein. We demonstrate that MLuc7 produced in insect cells is highly active and extremely thermostable, and is well suited as a secreted reporter when expressed in mammalian cells ensuring higher sensitivity of detection as compared to another Metridia luciferase isoform (MLuc164) which is widely employed in real-time imaging. (C) 2014 Elsevier Inc. All rights reserved.
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18.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Borisova V.V., Frank L.A., Markova S.V., Burakova L.P., Vysotski E.S.
Заглавие : Recombinant Metridia luciferase isoforms: expression, refolding and applicability for in vitro assay
Колич.характеристики :7 с
Коллективы :
Место публикации : Photochem. Photobiol. Sci.: ROYAL SOC CHEMISTRY, 2008. - Vol. 7, Is. 9. - С. 1025-1031. - ISSN 1474-905X, DOI 10.1039/b807271j
Примечания : Cited References: 19. - The work was supported by Bayer AG, by the Russian Foundation for Basic Research grants 05-04-48271 and 06-04-08076, by the joint grant 06-04-89502 of the Russian Foundation for Basic Research and Taiwan National Science Council, and by the "Molecular and Cellular Biology" program of the Russian Academy of Sciences.
Предметные рубрики: BIOLUMINESCENT REPORTER
GAUSSIA LUCIFERASE
CDNA
PROTEINS
CLONING
OVEREXPRESSION
PURIFICATION
MUTAGENESIS
ENZYME
OBELIN
Аннотация: The recombinant coelenterazine-dependent luciferases (isoforms MLuc 164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. coli cells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc09) was obtained in an active monomeric form with a high yield. The luciferase bioluminescence is found to be initiated not only by free coelenterazine, but also by Ca2+-dependent coelenterazine-binding protein (CBP) of Renilla muelleri on Ca2+ addition. The use of CBP as a "substrate" provides higher light emission and simultaneously the lower level of background. The high purity MLuc39 can be detected down to attomol with a linear range extending over 5 orders of magnitude. The MLuc39 reveals also a high stability towards heating and chemical modification; the chemically synthesized biotinylated derivatives of the luciferase preserve 35-40% of the initial activity The luciferase applicability as an in vitro bioluminescent reporter is demonstrated in model tandem bioluminescent solid-phase microassay combining the Ca2+-regulated photoprotein obelin and the Metridia luciferase.
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19.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Stepanyuk G.A., Xu H..., Wu C.K., Markova S.V., Lee J..., Vysotski E.S., Wang B.C.
Заглавие : Expression, purification and characterization of the secreted luciferase of the copepod Metridia longa from Sf9 insect cells
Колич.характеристики :7 с
Коллективы :
Место публикации : Protein Expr. Purif.: ACADEMIC PRESS INC ELSEVIER SCIENCE, 2008. - Vol. 61, Is. 2. - С. 142-148. - ISSN 1046-5928, DOI 10.1016/j.pep.2008.05.013
Примечания : Cited References: 34. - This work was supported by the National Institutes of Health (Grant 1P50 GM62407), University of Georgia Research Foundation and Georgia Research Alliance, the Russian Foundation for Basic Research and Taiwan National Science Council (Grant 06-0489502) and the program for "Molecular and Cellular Biology" of Russian Academy of Sciences.
Предметные рубрики: VARGULA-HILGENDORFII LUCIFERASE
CRYSTAL-STRUCTURE
RENILLA-RENIFORMIS
GAUSSIA LUCIFERASE
BIOLUMINESCENT REPORTER
OBELIN BIOLUMINESCENCE
ANGSTROM RESOLUTION
MAMMALIAN-CELLS
GENE-EXPRESSION
IN-VIVO
Аннотация: Metridia luciferase is a secreted luciferase from a marine copepod and uses coelenterazine as a substrate to produce a blue bioluminescence This luciferase has been successfully applied as a bioluminescent reporter in mammalian cells. The main advantage of secreted luciferase as a reporter is the capability of measuring intracellular events without destroying the cells or tissues and this property is well suited for development of high throughput screening technologies. However because Metridia luciferase is a Cys-rich protein, Escherichia coli expression systems produce an incorrectly folded protein, hindering its biochemical characterization and application for development of in vitro bioluminescent assays. Here we report the successful expression of Metridia luciferase with its signal peptide for secretion, in insect (Sf9) cells using the baculovirus expression system. Functionally active luciferase secreted by insect cells into the culture media has been efficiently purified with a yield of high purity protein of 2-3mg/L This Metridia luciferase expressed in the insect cell system is a monomeric protein showing 3.5-fold greater bioluminescence activity than luciferase expressed and purified from E. coli. The near coincidence of the experimental mass of Metridia luciferase purified from insect cells with that calculated from amino acid sequence. indicates that luciferase does not undergo post-translational modifications such as phosphorylation or glycosylation and also, the cleavage site of the signal peptide for secretion is at VQA-KS, as predicted from sequence analysis. (c) 2008 Elsevier Inc. All rights reserved.
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20.

Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Titushin M.S., Markova S.V., Frank L.A., Malikova N.P., Stepanyuk G.A., Lee J..., Vysotski E.S.
Заглавие : Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase
Колич.характеристики :8 с
Место публикации : Photochem. Photobiol. Sci.: ROYAL SOC CHEMISTRY, 2008. - Vol. 7, Is. 2. - С. 189-196. - ISSN 1474-905X, DOI 10.1039/b713109g
Примечания : Cited References: 41
Предметные рубрики: CRYSTAL-STRUCTURE
LIGHT-EMISSION
CA2+-REGULATED PHOTOPROTEINS
BIOLUMINESCENT REPORTER
RENIFORMIS LUCIFERASE
ANGSTROM RESOLUTION
RECOMBINANT OBELIN
ENERGY-TRANSFER
EXCITED-STATE
CALCIUM
Аннотация: The Renilla bioluminescent system in vivo is comprised of three proteins-the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca2+-binding superfamily of proteins with only three of the EF-hand loops having the Ca2+-binding consensus sequences. There is weak sequence homology with the Ca2+-regulated photoproteins but only as a result of the necessary Ca2+-binding loop structure. In combination with Renilla luciferase, addition of only one Ca2+ is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.
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