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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) : Bondar V.S., Purtov K.V., Malikova N.P., Frank L.A., Illarionov B.A.
Заглавие : Role of conservative residue Cys158 in the formation of an active photoprotein complex of obelin
Колич.характеристики :5 с
Место публикации : Biochem.-Moscow: MAIK NAUKA/INTERPERIODICA, 2001. - Vol. 66, Is. 9. - С. 1014-1018. - ISSN 0006-2979, DOI 10.1023/A:1012377827626
Примечания : Cited References: 21
Предметные рубрики: CDNA
EXPRESSION
AEQUORIN
SEQUENCE
CLONING
Ключевые слова (''Своб.индексиров.''): photoproteins--obelin--apoobelin mutants--bioluminescence
Аннотация: Using site directed mutagenesis, the conservative residue Cys158 of recombinant apoobelin was substituted for sera ine (C158S, S-mutant) or alanine (C158A, A-mutant). These point mutations resulted in significant changes in the apoobelin structure accompanied by slowing of photoprotein complex formation, decrease of its stability, and changing of its bioluminescence characteristics. The enzymatic properties of the photoprotein decreased in the series: wild-type protein S-mutant A-mutant. This is consistent with rank of nucleophilicity SH OH CH3 of cysteine, serine, and alanine side chain functional groups, respectively. Possible mechanisms of the involvement of the apoobelin Cys158 SH-group in the formation of the enzyme-substrate complex are considered.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Krasitskaya V. V., Burakova L. P., Komarova A. A., Bashmakova E. E., Frank L. A.
Заглавие : Mutants of Ca2+-regulated Photoprotein Obelin for Site-specific Conjugation
Место публикации : Photochem. Photobiol.: Blackwell Publishing Inc., 2017. - Vol. 93, Is. 2. - С. 553-557. - ISSN 00318655 (ISSN) , DOI 10.1111/php.12712
Аннотация: Color variants of Ca2+-regulated photoprotein obelin were shown to be an important tool for dual-analyte binding assay. To provide site-directed conjugation with biospecific molecules, several obelin color mutants carrying unique cysteine residues were obtained and characterized for their novel properties. A pair of obelins Y138F,A5C and W92F,H22E,D12C was found to be most suitable (in terms of high bioluminescent activity and stability) as reporters in simultaneous assay of two targets in a sample. Availability of SH-groups, accessible for chemical modification, essentially simplifies the synthesis of biospecific conjugates, increases their yield and conserves obelins' bioluminescence activity. Conjugates with immunoglobulin and oligonucleotide were produced and successfully applied in single nucleotide polymorphism genotyping. © 2017 The American Society of Photobiology
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Setkov N.A.
Заглавие : Liposomal cysteine proteinases inhibitors can commitment resting NIH 3T3 cells to proliferate
Колич.характеристики :4 с
Место публикации : Dokl. Akad. Nauk: MEZHDUNARODNAYA KNIGA, 1997. - Vol. 354, Is. 3. - P405-408. - ISSN 0869-5652
Примечания : Cited References: 14
Предметные рубрики: FIBROBLASTS
GROWTH
DEGRADATION
TURNOVER
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva, Elena V., Vysotski, Eugene S.
Заглавие : Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin
Колич.характеристики :9 с
Коллективы : Russian Academy of Sciences [03562016-0712, 0356-2015-0103]; RFBR [17-04-00764]
Место публикации : J. Photochem. Photobiol. B-Biol.: ELSEVIER SCIENCE SA, 2017. - Vol. 174. - С. 97-105. - ISSN 1011-1344, DOI 10.1016/j.jphotobio1.2017.07.021
Примечания : Cited References:54. - This work was supported by the state budget allocated to the fundamental research at the Russian Academy of Sciences (projects 03562016-0712 and 0356-2015-0103) and the RFBR grant 17-04-00764.
Предметные рубрики: SEQUENCE-ANALYSIS
APO-OBELIN
INTRINSIC FLUORESCENCE
COELENTERAZINE
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenterazine--photoprotein--coelenteramide--cysteine--serine
Аннотация: Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or p-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coil, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild -type aequorin. In contrast, Cys-free obelin retains only 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a "fast" component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3 0.2 and 44.6 0.4 C for aequorin and Cys-free aequorin, and 49.1 0.1 and 28.8 0.3 C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield.
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Вид документа : Статья из журнала
Шифр издания :
Автор(ы) : Eremeeva E. V., Vysotski E. S.
Заглавие : Bioluminescent and biochemical properties of Cys-free Ca2+-regulated photoproteins obelin and aequorin
Место публикации : J. Photochem. Photobiol. B Biol.: Elsevier B.V., 2017. - Vol. 174. - С. 97-105. - ISSN 10111344 (ISSN) , DOI 10.1016/j.jphotobiol.2017.07.021
Ключевые слова (''Своб.индексиров.''): bioluminescence--coelenteramide--coelenterazine--cysteine--photoprotein--serine
Аннотация: Bioluminescence of a variety of marine coelenterates is determined by Ca2+-regulated photoproteins. A strong interest in these proteins is for their wide analytical potential as intracellular calcium indicators and labels for in vitro binding assays. The presently known hydromedusan Ca2+-regulated photoproteins contain three (aequorin and clytin) or five (obelin and mitrocomin) cysteine residues with one of them strictly conserved. We have constructed Cys-free aequorin and obelin by substitution of all cysteines to serine residues. Such mutants should be of interest for researchers by the possibility to avoid the incubation with dithiothreitol (or ?-mercaptoethanol) required for producing an active photoprotein that is important for some prospective analytical assays in which the photoprotein is genetically fused with a target protein sensitive to the reducing agents. Cys-free mutants were expressed in Escherichia coli, purified, and characterized regarding the efficiency of photoprotein complex formation, functional activity, and conformational stability. The replacement of cysteine residues has been demonstrated to affect different properties of aequorin and obelin. Cys-free aequorin displays a two-fold lower specific bioluminescence activity but preserves similar activation properties and light emission kinetics compared to the wild-type aequorin. In contrast, Cys-free obelin retains only ~ 10% of the bioluminescence activity of wild-type obelin as well as binding coelenterazine and forming active photoprotein much less effectively. In addition, the substitution of Cys residues drastically changes the bioluminescence kinetics of obelin completely eliminating a “fast” component from the light signal decay curve. At the same time, the replacement of Cys residues increases conformational flexibility of both aequorin and obelin molecules, but again, the effect is more prominent in the case of obelin. The values of thermal midpoints of unfolding (Tm) were determined to be 53.3 ± 0.2 and 44.6 ± 0.4 °C for aequorin and Cys-free aequorin, and 49.1 ± 0.1 and 28.8 ± 0.3 °C for obelin and Cys-free obelin, respectively. Thus, so far only Cys-free aequorin is suitable as a partner for fusing with a tag sensitive to reducing agents since the aequorin mutant preserves almost 50% of the bioluminescent activity and can be produced with a substantial yield. © 2017 Elsevier B.V.
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