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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Markova, Svetlana V., Larionova, Marina D., Gorbunova, Darya A., Vysotski, Eugene S.
Заглавие : The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells
Колич.характеристики :7 с
Коллективы : RFBR; Government of Krasnoyarsk Territory [16-44-242099]; Russian Academy of Sciences [0356-2016-0712]
Место публикации : J. Photochem. Photobiol. B-Biol.: ELSEVIER SCIENCE SA, 2017. - Vol. 175. - С. 51-57. - ISSN 1011-1344, DOI 10.1016/j.jphotobiol.2017.08.024
Примечания : Cited References:30. - These studies were funded by RFBR and the Government of Krasnoyarsk Territory according to the research project No. 16-44-242099 and the state budget allocated to the fundamental research at the Russian Academy of Sciences (project No. 0356-2016-0712).
Предметные рубрики: GAUSSIA-PRINCEPS LUCIFERASE
ESCHERICHIA-COLI
EXPRESSION
PROTEIN
Ключевые слова (''Своб.индексиров.''): copepod luciferase--disulfide bonds--cysteine-rich protein--oxidative--refolding
Аннотация: The bioluminescence of a marine copepod Metridia Tonga is determined by a small secreted coelenterazine-dependent luciferase that uses coelenterazine as a substrate of enzymatic reaction to generate light (lambda(max) = 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 S-S 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. colt 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. coil 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.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Kirillova M. A., Ranjan R., Esimbekova E. N., Kratasyuk V. A.
Заглавие : Role of Hsp90 and ATP in modulating apyrase activity and firefly luciferase kinetics
Место публикации : Int. J. Biol. Macromol.: Elsevier B.V., 2019. - Vol. 131. - С. 691-696. - ISSN 01418130 (ISSN) , DOI 10.1016/j.ijbiomac.2019.03.110
Ключевые слова (''Своб.индексиров.''): bioluminescence--heat shock protein 90--high-throughput screening--adenosine triphosphate--apyrase--bovine serum albumin--firefly luciferase--heat shock protein 90--stabilizing agent--article--bioluminescence--clinical study--conformation--controlled study--denaturation--enzyme activity--enzyme kinetics--high throughput screening--incubation time--nonhuman--protein protein interaction--protein refolding--temperature--thermal denaturation--time
Аннотация: The present manuscript describes a novel bioassay consisting of apyrase and heat shock protein 90 (Hsp90) without additional co-chaperone supplementation; intended for high-throughput screening of anti-cancer drugs and prognosis of stress. In this regard, Hsp90 and adenosine 5?-triphosphate (ATP) mediated firefly luciferase (FLuc) kinetics was investigated using apyrase and FLuc as client proteins. Bioluminescent assay containing Hsp90, ATP, and apyrase led to complete loss of luminescence at 50 °C which indicates the protective role of Hsp90 against thermal denaturation. Similarly, the assay sample comprising Hsp90, ATP, and FLuc showed 2 fold increments in luminescence than their counterparts. Introduction of bovine serum albumin (BSA) to the pre-incubated assay mixture led to an initial rise in the luminescence (28%) in comparison to the sample containing Hsp90, ATP and FLuc. Therefore, FLuc based HTS assays are not suitable for clinical samples which may contain stabilizing agents. However, thermally denatured FLuc and apyrase could not regain their active conformation even when Hsp90 and ATP were introduced in the assay system. This observation justifies the role of Hsp90 to be protective rather than a reparation agent when acts without co-chaperones. © 2019
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Borisova V.V., Frank L.A., Markova S.V., Golz S..., Vysotski E.S.
Заглавие : Refolding of the recombinant luciferases of Metridia longa
Колич.характеристики :1 с
Место публикации : Luminescence: WILEY-BLACKWELL, 2006. - Vol. 21, Is. 5. - С. 271-271. - ISSN 1522-7235
Примечания : Cited References: 0
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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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Вид документа : Статья из сборника (однотомник)
Шифр издания :
Автор(ы) : Borisova V.V., Frank L.A., Markova S.V., Golz S..., Vysotski E.S.
Заглавие : REFOLDING OF THE RECOMBINANT LUCIFERASES OF METRIDIA LONGA
Колич.характеристики :4 с
Место публикации : BIOLUMINESCENCE AND CHEMILUMINESCENCE: CHEMISTRY, BIOLOGY AND APPLICATIONS: WORLD SCIENTIFIC PUBL CO PTE LTD, 2007. - 14th International Symposium on Bioluminescence and Chemiluminescence (OCT 15-19, 2006, San Diego, CA). - С. 3-6. - ISBN 978-981-270-816-8, DOI 10.1142/9789812770196_0001
Примечания : Cited References: 3
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