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Eugene S. Vysotski - One of the best experts on this subject based on the ideXlab platform.
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The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins.
International journal of molecular sciences, 2020Co-Authors: Marina Larionova, Svetlana V Markova, Nina V. Tikunova, Eugene S. VysotskiAbstract: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.
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The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.
Journal of photochemistry and photobiology. B Biology, 2017Co-Authors: Svetlana V Markova, Marina D Larionova, Darya A. Gorbunova, Eugene S. VysotskiAbstract:Abstract 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 (λ 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. 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.
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the smallest natural high active luciferase cloning and characterization of novel 16 5 kda luciferase from copepod Metridia longa
Biochemical and Biophysical Research Communications, 2015Co-Authors: Svetlana V Markova, Ludmila P Burakova, Marina D Larionova, Eugene S. VysotskiAbstract: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 SS 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 ∼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.
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high active truncated luciferase of copepod Metridia longa
Biochemical and Biophysical Research Communications, 2012Co-Authors: Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.
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recombinant Metridia luciferase isoforms expression refolding and applicability for in vitro assay
Photochemical and Photobiological Sciences, 2008Co-Authors: Vasilisa V Borisova, Ludmila A Frank, Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. colicells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) 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.
Tsutomu Ikeda - One of the best experts on this subject based on the ideXlab platform.
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Population structure, egg production and gut content pigment of large grazing copepods during the spring phytoplankton bloom in the Oyashio region
Deep-sea Research Part Ii-topical Studies in Oceanography, 2010Co-Authors: Atsushi Yamaguchi, Yuka Onishi, Momoka Kawai, Aya Omata, Mariko Kaneda, Tsutomu IkedaAbstract:As a basis for analyzing development of six large grazing copepods (Eucalanus bungii, Metridia pacifica, M. okhotensis, Neocalanus cristatus, N. flemingeri and N. plumchrus) in the Oyashio region, quasi-daily twin-NORPAC net (0.33 and 0.10 mm mesh) hauls were taken through the upper 150 m and 500 m at a station southwest of Hokkaido before (9–14 March) and after (6–30 April) the onset of the phytoplankton bloom in 2007. Based on additional fresh specimens collected from 0–150 m, egg production of E. bungii, M. pacifica and M. okhotensis, and gut pigments of late copepodid stages in each species were evaluated. Total zooplankton biomass was greater from 10 April onward by a factor of 2- to 8-fold the previous levels. This increase of the 0–150 m biomass was caused by development of Neocalanus spp. copepodids and upward migration of resting E. bungii. Egg production of E. bungii peaked on 18 April, while abundance of its nauplii and C1 peaked on 20 and 25 April, respectively. Sex ratio and C6-female gonad maturation index of E. bungii showed new recruitment to C6 during 20–30 April, likely derived from a population that over-wintered as C3 or C4. Egg production and hatchability of M. pacifica and M. okhotensis were highly variable and no temporal trend was detected. Comparison with field abundance data for Metridia spp. suggests that our estimates of egg production and hatchability are too low, despite care with experimental conditions. All the Neocalanus species utilize the bloom as energy for juvenile growth. Neocalanus cristatus developed from C2 through C4, and stage duration of C3 was estimated to be 24 days. Neocalanus flemingeri also developed from C1 through C3, and stage durations of C1 and C2 were estimated to be 7–9 days. Neocalanus plumchrus occurred in small numbers from mid-April onward. The stage duration estimates for Neocalanus spp. are similar to those reported from the high-nutrition southeastern Bering Sea shelf. Gut pigment variation clearly showed nocturnal feeding by Metridia spp., while no diel changes in gut pigment were recognized for E. bungii or Neocalanus spp. The diel changes in gut pigment of Metridia spp. were related to their diel vertical migrations. The calendar of sequential responses of copepods to the phytoplankton bloom is summarized.
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Diel and ontogenetic variations in vertical distributions of large grazing copepods during the spring phytoplankton bloom in the Oyashio region
Deep Sea Research Part II: Topical Studies in Oceanography, 2010Co-Authors: Atsushi Yamaguchi, Yuka Onishi, Momoka Kawai, Aya Omata, Mariko Kaneda, Tsutomu IkedaAbstract:Short-term changes in vertical distributions of copepods during the spring phytoplankton bloom were analyzed based on day and night vertically stratified sampling (9 strata between 0-1000 m) with a fine-mesh (60 μm) VMPS in the Oyashio region on five occasions: 8 March, 5, 11, 23 and 29 April 2007. Responses to the bloom were varied and species-specific. Eucatanus bungii C3-C6 were resting around 400 m on 8 March. They had aroused from rest by 5 April, more completely for C6F than for C3-C4. On 29 April, newly recruited C1-C4 stayed in near surface layers (0-50 m). Both Metridia pacifica and Metridia okhotensis showed strong diel vertical migrations (DVM). When the amount of sinking flux was sufficient (23 and 29 April), juveniles ceased DVM and stayed close to 300 m throughout the day and night, while the C6F continued DVM. Continuous DVM behavior of Metridia spp. C6F is likely related to spawning in the surface layer at night. The growth phase of juvenile Neocalanus spp. occurred shallower than 250 m. Within this depth range, vertical partitioning was observed among the species: Neocalanus flemingeri and Neocalanus plumchrus occurred above 50 m, while Neocalanus cristatus was distributed from 75-250 m. The boundary between two patterns was around 50-75 m. These findings are comparable to those in the eastern subarctic Pacific
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Ecological features of meso- and bathypelagic copepods as viewed from prey-predator interactions
2003Co-Authors: Atsushi Yamaguchi, Tsutomu IkedaAbstract:Recent progress in the study on vertical partitioning, ontogenetic vertical migration and life cycles of meso- and bathypelagic copepods in the western subarctic Pacific is reviewed from the viewpoint of prey-predator interactions. Gaidius variabilis and Pleuromamma scutullata are particle feeders, and their spawning seasons were well synchronized with that of the phytoplankton bloom. Judging from their vertical distribution depth (G. variabilis; 600-1500 m, P. scutullata; 200-600 m), it is suggested that they are depending on directly sinking particles from the euphotic zone. Scolecithricella minor is known to be a detritus feeder, and their reproduction/active growth season was closely associated with high abundance of appendiculari- ans. It implies that discarded 'house' of appendicularians is a major food source for S. minor. As a typical carnivore, Heterorhabdus tanneri is characterized by intermolt growth of young copepodids being 3 times greater than those of other species, and they achieve a langeorganicmatter deposition during high the abundance season of their potential food (other zooplankton). The vertical distribution of congener species is never overlapped. In the subarctic Pacific, four Metridia spp. and three Paraeuchaeta spp. are predominant. For Metridia species, M pacifica distributed mainly at 0-250 m, M okhotensis at 250-1000 m, M asymmetrica at 1000-2000 m and M curticauda at >2000 m depths. For Paraeuchaeta, P. elongata was at 0-500 m, P. birostrata at 250-1000 m and P. rubra at 500-2000 m depths. Since feeding modes of congener species are similar, vertical partitioning of their habitats within the genus is advantageous to reduce feeding competition between the species. The ontogenetic vertical migration patterns of meso- and bathypelagic copepods are characterized by developmental ascent. In the subarctic Pacific, three Paraeuchaeta species, Gaidius variabilis and Metridia asymmetrica exhibited the ontogenetic vertical migration pattern. Since their lifetime fecundity is low, starting the early life at depth is advantageous to reduce their predation mortalities. This explanation appears to be consistent with the observation that the fecundity of these copepods decreases with increasing depth. At the same time, developmental ascent is considered to be advantageous to achieve better growth in food-rich upper layers.
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of lowered pH to some oceanic zooplankton
1999Co-Authors: Yuichiro Yamada, Tsutomu IkedaAbstract:Acute toxicity of lowered pH (s4) was tested on 10 oceanic zooplankton species, including Conchoecia sp., Calanus pacificus, Neocalanus cristatus, Eucalanus bungii bungii, Pseudocalanus minutus, Metridia pacifica, Paraeuchaeta elongata, Themisto japonica, Euphausia pacifica (nauplii and juveniles) and Sagitta elegans. As indices of pH sensitivity in the zooplankton, the pH levels caus ing 50% mortality (LC50) and zero mortality (LC0) were estimated from the mortality-log10( pH) rela
Gerd-peter Zauke - One of the best experts on this subject based on the ideXlab platform.
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Trace metals in Antarctic copepods from the Weddell Sea (Antarctica).
Chemosphere, 2003Co-Authors: J. Kahle, Gerd-peter ZaukeAbstract:The concentrations of Cd, Co, Cu, Ni, Pb and Zn were determined in the Antarctic copepods Rhincalanus gigas (Brady, 1883), Calanus propinquus (Brady, 1883), Calanoides acutus (Giesbrecht, 1902), Metridia curticauda (Giesbrecht, 1889) and Metridia gerlachei (Giesbrecht, 1902). Samples were taken at seven different stations between 18.01.1999 and 19.02.1999. Metal concentrations in biological tissue were determined by graphite furnace atomic absorption spectroscopy (GFAAS) with Zeeman background correction and by flame AAS (air-acetylene) with deuterium background correction. We found high mean Cd concentrations in the Metridia species of about 10 microg Cd g(-1) and 3-6 microg Cd g(-1) in the other copepods. Co and Pb concentrations were low in all species investigated (
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Bioaccumulation of trace metals in the calanoid copepod Metridia gerlachei from the Weddell Sea (Antarctica).
Science of The Total Environment, 2002Co-Authors: J. Kahle, Gerd-peter ZaukeAbstract:Abstract Bioaccumulation of Cd, Co, Cu, Ni, Pb and Zn in the Antarctic calanoid copepod Metridia gerlachei (Giesbrecht 1902) was investigated during a cruise of RV ‘ Polarstern ’ to the Weddell Sea, primarily to provide information on accumulation strategies for the metals tested. With the sole exception of Cd, the copepod accumulated metals during exposure and depurated them in uncontaminated seawater. The process of uptake and depuration was successfully described by a hyperbolic model, leading to significant estimations of the following experimental bioconcentration factors (BCFs): 210 (Co), 3430 (Cu), 3060 (Ni), 670 (Pb) and 2090 (Zn). Furthermore, we provide an approach to evaluate the sensitivity of Metridia gerlachei as a biomonitor of water-borne metals in the field; the results indicate minimal increments in ambient exposure concentrations of: 0.5 μg Cu l −1 , 0.8 μg Ni l −1 , 0.6 μg Pb l −1 and 0.2 μg Zn l −1 , suggesting a high sensitivity of M. gerlachei for biomonitoring.
Atsushi Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
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Seasonal changes in the population structure of dominant planktonic copepods collected using a sediment trap moored in the western Arctic Ocean
Journal of Natural History, 2015Co-Authors: Kohei Matsuno, Atsushi Yamaguchi, Amane Fujiwara, Jonaotaro Onodera, Eiji Watanabe, Naomi Harada, Takashi KikuchiAbstract:Winter ice cover of the Arctic Ocean makes year-round zooplankton sampling by plankton net a difficult task. Therefore, the collection of copepods with a sediment trap can be a powerful tool. In the present study, we analysed the seasonal changes in the population structures of five dominant planktonic copepods (Oncaea parila, Calanus hyperboreus, Metridia longa, Paraeuchaeta glacialis and Heterorhabdus norvegicus), which were collected using a sediment trap rotated at 10–15 day intervals moored at 184–260 m in the Northwind Abyssal Plain (75°00′N, 162°00′W) of the western Arctic Ocean from October 2010 to September 2012. Oncaea parila C6F with egg sacs occurred throughout the year, and the total abundance and composition of early copepodid stages (C1−C3) had two peaks each year. Calanus hyperboreus was dominated by C6F throughout the year, and their maturation was observed during February to May. Metridia longa C6F had a clear seasonality in lipid accumulation and gonad maturation: high lipid accumulatio...
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SEASONAL AND INTER-SPECIES COMPARISON OF ASYMMETRY IN THE GENITAL SYSTEM OF SOME SPECIES OF THE OCEANIC COPEPOD GENUS Metridia (COPEPODA, CALANOIDA)
Crustaceana, 2015Co-Authors: Daichi Arima, Atsushi Yamaguchi, Kohei Matsuno, Takahiro Nobetsu, Ichiro ImaiAbstract:The seasonal and inter-annual changes in the asymmetry of female insemination and the male leg 5 of the planktonic calanoid copepods Metridia okhotensis and M. pacifica were investigated in the Okhotsk Sea. An inter-species comparison of both parameters was also carried out on seven Metridia species collected from oceans throughout the world. For M. okhotensis from the Okhotsk Sea, most of the females showed left-side insemination (annual average: 95.7%) and most of the males showed left-side asymmetry (99.7%) of the long inner process of the second exopodal segment in the fifth leg throughout the year. However, sympatric M. pacifica showed different ratios of asymmetry for female insemination and male morphotypes with a left : right = 1 : 2 ratio throughout the year. For the seven Metridia species from the global oceans treated in this study, ratios of asymmetry for female insemination and male morphotypes were correlated with each other. One-sided insemination (i.e., only left or only right insemination) was a common pattern for various Metridia species from global oceans, but their ratios varied by species. Previously, low hatching rates (29-68%) of eggs were reported for various Metridia spp. in laboratory experiments, but the reasons for these low rates were unclear. Because each spermatheca of Metridia spp. is connected to the oviduct on the same side, either left or right, this suggests that half of the eggs produced by unilaterally inseminated females remain unfertilized. The morphology of the genital structures and literature data of the egg hatching rates of Metridia spp. indicate that almost half of the eggs produced by females are not viable and are, thus, wasted.
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Computational analysis and functional expression of ancestral copepod luciferase.
Gene, 2013Co-Authors: Yasuhiro Takenaka, Akiko Noda-ogura, Tadashi Imanishi, Takashi Gojobori, Atsushi Yamaguchi, Yasushi ShigeriAbstract:Abstract We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species (Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species.
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Population structure, egg production and gut content pigment of large grazing copepods during the spring phytoplankton bloom in the Oyashio region
Deep-sea Research Part Ii-topical Studies in Oceanography, 2010Co-Authors: Atsushi Yamaguchi, Yuka Onishi, Momoka Kawai, Aya Omata, Mariko Kaneda, Tsutomu IkedaAbstract:As a basis for analyzing development of six large grazing copepods (Eucalanus bungii, Metridia pacifica, M. okhotensis, Neocalanus cristatus, N. flemingeri and N. plumchrus) in the Oyashio region, quasi-daily twin-NORPAC net (0.33 and 0.10 mm mesh) hauls were taken through the upper 150 m and 500 m at a station southwest of Hokkaido before (9–14 March) and after (6–30 April) the onset of the phytoplankton bloom in 2007. Based on additional fresh specimens collected from 0–150 m, egg production of E. bungii, M. pacifica and M. okhotensis, and gut pigments of late copepodid stages in each species were evaluated. Total zooplankton biomass was greater from 10 April onward by a factor of 2- to 8-fold the previous levels. This increase of the 0–150 m biomass was caused by development of Neocalanus spp. copepodids and upward migration of resting E. bungii. Egg production of E. bungii peaked on 18 April, while abundance of its nauplii and C1 peaked on 20 and 25 April, respectively. Sex ratio and C6-female gonad maturation index of E. bungii showed new recruitment to C6 during 20–30 April, likely derived from a population that over-wintered as C3 or C4. Egg production and hatchability of M. pacifica and M. okhotensis were highly variable and no temporal trend was detected. Comparison with field abundance data for Metridia spp. suggests that our estimates of egg production and hatchability are too low, despite care with experimental conditions. All the Neocalanus species utilize the bloom as energy for juvenile growth. Neocalanus cristatus developed from C2 through C4, and stage duration of C3 was estimated to be 24 days. Neocalanus flemingeri also developed from C1 through C3, and stage durations of C1 and C2 were estimated to be 7–9 days. Neocalanus plumchrus occurred in small numbers from mid-April onward. The stage duration estimates for Neocalanus spp. are similar to those reported from the high-nutrition southeastern Bering Sea shelf. Gut pigment variation clearly showed nocturnal feeding by Metridia spp., while no diel changes in gut pigment were recognized for E. bungii or Neocalanus spp. The diel changes in gut pigment of Metridia spp. were related to their diel vertical migrations. The calendar of sequential responses of copepods to the phytoplankton bloom is summarized.
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Diel and ontogenetic variations in vertical distributions of large grazing copepods during the spring phytoplankton bloom in the Oyashio region
Deep Sea Research Part II: Topical Studies in Oceanography, 2010Co-Authors: Atsushi Yamaguchi, Yuka Onishi, Momoka Kawai, Aya Omata, Mariko Kaneda, Tsutomu IkedaAbstract:Short-term changes in vertical distributions of copepods during the spring phytoplankton bloom were analyzed based on day and night vertically stratified sampling (9 strata between 0-1000 m) with a fine-mesh (60 μm) VMPS in the Oyashio region on five occasions: 8 March, 5, 11, 23 and 29 April 2007. Responses to the bloom were varied and species-specific. Eucatanus bungii C3-C6 were resting around 400 m on 8 March. They had aroused from rest by 5 April, more completely for C6F than for C3-C4. On 29 April, newly recruited C1-C4 stayed in near surface layers (0-50 m). Both Metridia pacifica and Metridia okhotensis showed strong diel vertical migrations (DVM). When the amount of sinking flux was sufficient (23 and 29 April), juveniles ceased DVM and stayed close to 300 m throughout the day and night, while the C6F continued DVM. Continuous DVM behavior of Metridia spp. C6F is likely related to spawning in the surface layer at night. The growth phase of juvenile Neocalanus spp. occurred shallower than 250 m. Within this depth range, vertical partitioning was observed among the species: Neocalanus flemingeri and Neocalanus plumchrus occurred above 50 m, while Neocalanus cristatus was distributed from 75-250 m. The boundary between two patterns was around 50-75 m. These findings are comparable to those in the eastern subarctic Pacific
Svetlana V Markova - One of the best experts on this subject based on the ideXlab platform.
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The Smallest Isoform of Metridia longa Luciferase as a Fusion Partner for Hybrid Proteins.
International journal of molecular sciences, 2020Co-Authors: Marina Larionova, Svetlana V Markova, Nina V. Tikunova, Eugene S. VysotskiAbstract: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.
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The disulfide-rich Metridia luciferase refolded from E. coli inclusion bodies reveals the properties of a native folded enzyme produced in insect cells.
Journal of photochemistry and photobiology. B Biology, 2017Co-Authors: Svetlana V Markova, Marina D Larionova, Darya A. Gorbunova, Eugene S. VysotskiAbstract:Abstract 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 (λ 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. 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.
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the smallest natural high active luciferase cloning and characterization of novel 16 5 kda luciferase from copepod Metridia longa
Biochemical and Biophysical Research Communications, 2015Co-Authors: Svetlana V Markova, Ludmila P Burakova, Marina D Larionova, Eugene S. VysotskiAbstract: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 SS 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 ∼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.
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high active truncated luciferase of copepod Metridia longa
Biochemical and Biophysical Research Communications, 2012Co-Authors: Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The technology of real-time imaging in living cells is crucial for understanding of intracellular events. For this purpose, bioluminescent reporters have been introduced as sensitive and convenient tools. Metridia luciferase (MLuc) from the copepod Metridia longa is a coelenterazine-dependent luciferase containing a natural signal peptide for secretion. We report the high-active MLuc mutants with deletion of the N-terminal variable part of amino acid sequence. The MLuc variants were produced in Escherichia coli cells, converted to an active protein, and characterized. We demonstrate that the truncated MLucs have significantly increased bioluminescent activity as against the wild type enzyme but substantially retain other properties. One of the truncated variants of MLuc was transiently expressed in HEK 293 cells. The results clearly suggest that the truncated Metridia luciferase is well suited as a secreted reporter ensuring higher detection sensitivity in comparison with a wild type enzyme.
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recombinant Metridia luciferase isoforms expression refolding and applicability for in vitro assay
Photochemical and Photobiological Sciences, 2008Co-Authors: Vasilisa V Borisova, Ludmila A Frank, Svetlana V Markova, Ludmila P Burakova, Eugene S. VysotskiAbstract:The recombinant coelenterazine-dependent luciferases (isoforms MLuc164 and MLuc39) from the marine copepod Metridia longa were expressed as inclusion bodies in E. colicells, dissolved in 6 M guanidinium chloride and folded in conditions developed for proteins containing intramolecular disulfide bonds. One of them (MLuc39) 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.