LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein

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LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein (en)
Authors

Publications

Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli

Savić, Aleksa

(2022)

TY  - THES
AU  - Savić, Aleksa
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5566
AB  - Alfa-sinuklein je protein zastupljen u nervnom tkivu, dominantno u završecima presinaptičih neurona. Agregati ovog proteina pronađeni su u nervnom tkivu brojnih pacijenata sa bolestima koje se pod jednim imenom označavaju α-sinukleinopatijama, a od kojih je najznačajnija Parkinsonova bolest. Formiranje ovih agregata smatra se usko povezanim sa etiologijom ove bolesti i zbog toga je značajno proizvesti i prečistiti ovaj protein u velikim količinama u cilju in vitro, a potencijalno i in vivo istraživanja. Koristeći komercijalni vektor pDUET-1-α-synuclein-mCerulean3-His6, u našoj laboratoriji su prethodnim radom proizvedena tri vektora za ekspresiju α-sinukleina u bakteriji Escherichia coli. U ovom radu je opisana optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina sa sva tri plazmida transformisanih u sojeve E. coli BL21(DE3), odnosno BL21(DE3)pLysS, a koristeći metodologiju odzivne površine (RSM). Kombinacija plazmida i soja koja je pokazala najveći nivo ekspresije upotrebljena je za ekspresiju proteina od interesa na velikoj skali. Takođe, pokazano je da se dobijeni himerni protein nakon prečišćavanja imobilizovanom metal-afinitetnom hromatografijom može specifično hidrolizovati TEV proteazom. Nakon toga, α-sinuklein nativne sekvence dobijen proteolizom je grubo prečisćen ponovljenom metal-afinitetnom hromatografijom. Pokazano je i da prilikom zagrevanja himernog proteina ne dolazi do njegove značajne denaturacije i taloženja, što se može upotrebiti za postizanje značajnog prečišćenja jednim dodatnim korakom.
T1  - Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli
SP  - 1
EP  - 84
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5566
ER  - 
@mastersthesis{
author = "Savić, Aleksa",
year = "2022",
abstract = "Alfa-sinuklein je protein zastupljen u nervnom tkivu, dominantno u završecima presinaptičih neurona. Agregati ovog proteina pronađeni su u nervnom tkivu brojnih pacijenata sa bolestima koje se pod jednim imenom označavaju α-sinukleinopatijama, a od kojih je najznačajnija Parkinsonova bolest. Formiranje ovih agregata smatra se usko povezanim sa etiologijom ove bolesti i zbog toga je značajno proizvesti i prečistiti ovaj protein u velikim količinama u cilju in vitro, a potencijalno i in vivo istraživanja. Koristeći komercijalni vektor pDUET-1-α-synuclein-mCerulean3-His6, u našoj laboratoriji su prethodnim radom proizvedena tri vektora za ekspresiju α-sinukleina u bakteriji Escherichia coli. U ovom radu je opisana optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina sa sva tri plazmida transformisanih u sojeve E. coli BL21(DE3), odnosno BL21(DE3)pLysS, a koristeći metodologiju odzivne površine (RSM). Kombinacija plazmida i soja koja je pokazala najveći nivo ekspresije upotrebljena je za ekspresiju proteina od interesa na velikoj skali. Takođe, pokazano je da se dobijeni himerni protein nakon prečišćavanja imobilizovanom metal-afinitetnom hromatografijom može specifično hidrolizovati TEV proteazom. Nakon toga, α-sinuklein nativne sekvence dobijen proteolizom je grubo prečisćen ponovljenom metal-afinitetnom hromatografijom. Pokazano je i da prilikom zagrevanja himernog proteina ne dolazi do njegove značajne denaturacije i taloženja, što se može upotrebiti za postizanje značajnog prečišćenja jednim dodatnim korakom.",
title = "Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli",
pages = "1-84",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5566"
}
Savić, A.. (2022). Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli. , 1-84.
https://hdl.handle.net/21.15107/rcub_cherry_5566
Savić A. Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli. 2022;:1-84.
https://hdl.handle.net/21.15107/rcub_cherry_5566 .
Savić, Aleksa, "Optimizacija uslova ekspresije fluorescentno obeleženog humanog α-sinukleina u bakteriji Escherichia coli" (2022):1-84,
https://hdl.handle.net/21.15107/rcub_cherry_5566 .

Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects

Vidović, Marija; Battisti, Ilaria; Pantelić, Ana; Morina, Filis; Arrigoni, Giorgio; Masi, Antonio; Veljović Jovanović, Sonja

(2022)

TY  - CONF
AU  - Vidović, Marija
AU  - Battisti, Ilaria
AU  - Pantelić, Ana
AU  - Morina, Filis
AU  - Arrigoni, Giorgio
AU  - Masi, Antonio
AU  - Veljović Jovanović, Sonja
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5560
AB  - Ramonda serbica Panc. is a resurrection plant species that can survive desiccation for a long
period and fully resume metabolic functions upon rewatering in a very short period, even within
48 h. The goal of this study was to identify key candidates and pathways involved in desiccation tolerance in R. serbica. To achieve this, systems biology approach combining transcriptomics,
proteomics, and analysis of specific metabolites was employed. In addition, FTIR analysis of the
cell wall polymers and a detailed analysis of the photosynthetic electron transport (PET) chain
were performed. In total, 1192 different protein groups were quantified by TMT-based comparative quantitative proteomics. Among them, 408 protein groups showed a statistically significant
difference in abundance between hydrated (HL) and desiccated leaves (DL). Upon desiccation, the
majority of proteins related to photosynthetic processes were less abundant, while chlorophyll
fluorescence measurements implied shifting from linear photosynthetic transport (PET) to cyclic
electron transport (CET). The amounts of H2O2 scavenging enzymes, including ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn- superoxide dismutase (SOD) were
reduced in DL. However, four Cu/ZnSOD isoforms, three polyphenol oxidases, six germin-like proteins (GLPs), and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins),
were desiccation-inducible. Desiccation-induced cell wall remodelling by changes in cell wall
polymer composition might be linked with pectin demethylesterification and GLP-derived H2O2/
HO•. Our study demonstrated that desiccation tolerance in R. serbica is a complex, species-specific process orchestrated by several metabolic pathways and regulatory networks acting at the transcript, protein, metabolite and physiological levels.
C3  - 4th International Conference on Plant Biology, Book of Abstracts
T1  - Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects
SP  - 27
EP  - 27
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5560
ER  - 
@conference{
author = "Vidović, Marija and Battisti, Ilaria and Pantelić, Ana and Morina, Filis and Arrigoni, Giorgio and Masi, Antonio and Veljović Jovanović, Sonja",
year = "2022",
abstract = "Ramonda serbica Panc. is a resurrection plant species that can survive desiccation for a long
period and fully resume metabolic functions upon rewatering in a very short period, even within
48 h. The goal of this study was to identify key candidates and pathways involved in desiccation tolerance in R. serbica. To achieve this, systems biology approach combining transcriptomics,
proteomics, and analysis of specific metabolites was employed. In addition, FTIR analysis of the
cell wall polymers and a detailed analysis of the photosynthetic electron transport (PET) chain
were performed. In total, 1192 different protein groups were quantified by TMT-based comparative quantitative proteomics. Among them, 408 protein groups showed a statistically significant
difference in abundance between hydrated (HL) and desiccated leaves (DL). Upon desiccation, the
majority of proteins related to photosynthetic processes were less abundant, while chlorophyll
fluorescence measurements implied shifting from linear photosynthetic transport (PET) to cyclic
electron transport (CET). The amounts of H2O2 scavenging enzymes, including ascorbate-glutathione cycle components, catalases, peroxiredoxins, Fe-, and Mn- superoxide dismutase (SOD) were
reduced in DL. However, four Cu/ZnSOD isoforms, three polyphenol oxidases, six germin-like proteins (GLPs), and 22 late embryogenesis abundant proteins (LEAPs; mainly LEA4 and dehydrins),
were desiccation-inducible. Desiccation-induced cell wall remodelling by changes in cell wall
polymer composition might be linked with pectin demethylesterification and GLP-derived H2O2/
HO•. Our study demonstrated that desiccation tolerance in R. serbica is a complex, species-specific process orchestrated by several metabolic pathways and regulatory networks acting at the transcript, protein, metabolite and physiological levels.",
journal = "4th International Conference on Plant Biology, Book of Abstracts",
title = "Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects",
pages = "27-27",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5560"
}
Vidović, M., Battisti, I., Pantelić, A., Morina, F., Arrigoni, G., Masi, A.,& Veljović Jovanović, S.. (2022). Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects. in 4th International Conference on Plant Biology, Book of Abstracts, 27-27.
https://hdl.handle.net/21.15107/rcub_cherry_5560
Vidović M, Battisti I, Pantelić A, Morina F, Arrigoni G, Masi A, Veljović Jovanović S. Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects. in 4th International Conference on Plant Biology, Book of Abstracts. 2022;:27-27.
https://hdl.handle.net/21.15107/rcub_cherry_5560 .
Vidović, Marija, Battisti, Ilaria, Pantelić, Ana, Morina, Filis, Arrigoni, Giorgio, Masi, Antonio, Veljović Jovanović, Sonja, "Mechanisms of desiccation tolerance in Ramonda serbica Panc.: transcriptomic, proteomic, metabolomic, and photosynthetic aspects" in 4th International Conference on Plant Biology, Book of Abstracts (2022):27-27,
https://hdl.handle.net/21.15107/rcub_cherry_5560 .

Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves

Pantelić, Ana; Stevanovic, Strahinja; Milic-Komic, Sonja; Kilibarda, Natasa; Vidović, Marija

(MDPI, 2022)

TY  - DATA
AU  - Pantelić, Ana
AU  - Stevanovic, Strahinja
AU  - Milic-Komic, Sonja
AU  - Kilibarda, Natasa
AU  - Vidović, Marija
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/4882
AB  - Ramonda serbica de novo transcriptome database
PB  - MDPI
T2  - International Journal of Molecular Science
T1  - Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves
VL  - n/a
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4882
ER  - 
@misc{
author = "Pantelić, Ana and Stevanovic, Strahinja and Milic-Komic, Sonja and Kilibarda, Natasa and Vidović, Marija",
year = "2022",
abstract = "Ramonda serbica de novo transcriptome database",
publisher = "MDPI",
journal = "International Journal of Molecular Science",
title = "Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves",
volume = "n/a",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4882"
}
Pantelić, A., Stevanovic, S., Milic-Komic, S., Kilibarda, N.,& Vidović, M.. (2022). Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves. in International Journal of Molecular Science
MDPI., n/a.
https://hdl.handle.net/21.15107/rcub_cherry_4882
Pantelić A, Stevanovic S, Milic-Komic S, Kilibarda N, Vidović M. Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves. in International Journal of Molecular Science. 2022;n/a.
https://hdl.handle.net/21.15107/rcub_cherry_4882 .
Pantelić, Ana, Stevanovic, Strahinja, Milic-Komic, Sonja, Kilibarda, Natasa, Vidović, Marija, "Ramonda serbica de novo transcriptome database related to the article: Pantelic, A.; Stevanović, S.; Milic-Komic, S.; Kilibarda, N.; Vidovic, M. Characterization and expression analysis of the late embryogenesis abundant (LEA) proteins family in hydrated and desiccated Ramonda serbica Panc. leaves" in International Journal of Molecular Science, n/a (2022),
https://hdl.handle.net/21.15107/rcub_cherry_4882 .

Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli

Savić, Aleksa D.; Vidović, Marija; Radosavljević, Jelena

(Beograd : Srpsko hemijsko društvo, 2022)

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija
AU  - Radosavljević, Jelena
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5373
AB  - Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom.
AB  - Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.
PB  - Beograd : Srpsko hemijsko društvo
C3  - 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
T1  - Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli
T1  - Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli
SP  - 68
EP  - 68
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5373
ER  - 
@conference{
author = "Savić, Aleksa D. and Vidović, Marija and Radosavljević, Jelena",
year = "2022",
abstract = "Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine",
title = "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli, Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli",
pages = "68-68",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5373"
}
Savić, A. D., Vidović, M.,& Radosavljević, J.. (2022). Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
Beograd : Srpsko hemijsko društvo., 68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5373
Savić AD, Vidović M, Radosavljević J. Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine. 2022;:68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5373 .
Savić, Aleksa D., Vidović, Marija, Radosavljević, Jelena, "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli" in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine (2022):68-68,
https://hdl.handle.net/21.15107/rcub_cherry_5373 .

Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli

Savić, Aleksa D.; Vidović, Marija; Radosavljević, Jelena

(Beograd : Srpsko hemijsko društvo, 2022)

TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija
AU  - Radosavljević, Jelena
PY  - 2022
UR  - http://cherry.chem.bg.ac.rs/handle/123456789/5374
AB  - Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom.
AB  - Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.
PB  - Beograd : Srpsko hemijsko društvo
C3  - 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
T1  - Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli
T1  - Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli
SP  - 68
EP  - 68
UR  - https://hdl.handle.net/21.15107/rcub_cherry_5374
ER  - 
@conference{
author = "Savić, Aleksa D. and Vidović, Marija and Radosavljević, Jelena",
year = "2022",
abstract = "Fluorescentno obeleženi proteini su neprocenjivi alati u laboratorijskoj praksi za in vivo lokalizovanje i ispitivanje interakcija proteina. Dizajnirali smo vektor za ekspresiju mCerulean3 sa N-terminalnim heksahistidinskim obeleživacem fuzionisanim preko poliasparaginskog linkera i proteolitickog mesta za proteazu virusa graviranosti duvana (TEV) sa -sinukleinom. Ovaj konstrukt može se upotrebiti za proizvodnju -sinukleina nativne sekvence nakon proteolize TEV proteazom. Gen za mCerulean3 je nizom lancanih reakcija polimeraze (SOEing PCR) fuzionisan sa genom za -sinuklein i nakon amplifikacije ukloniran u plazmid pDUET-1. Escherichia coli BL21(DE3) je, nakon transformacije ovim konstruktom, upotrebljena za proizvodnju himernog proteina koji je zadržao fluorescentna svojstva sa prinosom od ~2 mg po litru medijuma nakon precišcavanja imobilizovanom metal-afinitetnom hromatografijom (elektroforetska cistoca: ~80%). Ovaj himerni protein je uspešno proteolizovan TEV proteazom., Fluorescently labeled proteins are invaluable tools in laboratory practice to assess the in vivo localization and the interactions of proteins. Here we have designed an expression vector with an N-terminal hexahistidine-tagged mCerulean3 fused through a polyasparagine linker and the proteolytic site of tobacco etch virus protease (TEV) to - synuclein. This construct can be used to produce -synuclein of a native sequence after proteolysis with TEV protease. After fusion of the genes for mCerulean3 and -synuclein through a series of polymerase chain reactions (SOEing PCR), the resulting gene for the chimeric protein was cloned into the pDUET-1 plasmid. Escherichia coli BL21(DE3), upon transformation with this construct, can be used to produce the chimeric protein that retained the fluorescent properties of mCerulean3, with a yield of ~2 mg per liter of medium after purification by immobilized metal-affinity chromatography (electrophoretic purity: ~80%). This chimeric protein was successfully proteolyzed by TEV protease.",
publisher = "Beograd : Srpsko hemijsko društvo",
journal = "58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine",
title = "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli, Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli",
pages = "68-68",
url = "https://hdl.handle.net/21.15107/rcub_cherry_5374"
}
Savić, A. D., Vidović, M.,& Radosavljević, J.. (2022). Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine
Beograd : Srpsko hemijsko društvo., 68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5374
Savić AD, Vidović M, Radosavljević J. Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli. in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine. 2022;:68-68.
https://hdl.handle.net/21.15107/rcub_cherry_5374 .
Savić, Aleksa D., Vidović, Marija, Radosavljević, Jelena, "Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli" in 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine (2022):68-68,
https://hdl.handle.net/21.15107/rcub_cherry_5374 .

De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins

Pantelić, Ana; Stevanović, Strahinja; Kilibarda, Nataša; Vidović, Marija

(Novi Sad : Faculty of Sciences, Department of Biology and Ecology, 2021)

TY  - CONF
AU  - Pantelić, Ana
AU  - Stevanović, Strahinja
AU  - Kilibarda, Nataša
AU  - Vidović, Marija
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4503
UR  - http://ojs.pmf.uns.ac.rs/index.php/dbe_serbica/index
AB  - An extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation, aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding, ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation.
Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs were classification into six groups according to Protein family (PFAM) database and the most populated group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence length is predicted to form α-helical structure). This implies that charged residues might be exposed to the solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in the cell. In addition, as predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered (~64%). Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.
PB  - Novi Sad : Faculty of Sciences, Department of Biology and Ecology
C3  - Biologia Serbica
T1  - De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins
VL  - 43
IS  - 1 (spec. ed.)
SP  - 65
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4503
ER  - 
@conference{
author = "Pantelić, Ana and Stevanović, Strahinja and Kilibarda, Nataša and Vidović, Marija",
year = "2021",
abstract = "An extreme loss of cellular water or desiccation (5-10% of relative water content) leads to protein denaturation, aggregation and degradation, and affects the fluidity of membrane lipids resulting in loss of membrane integrity [1]. The essential constituents of vegetative desiccation tolerance in so-called resurrection plants are late embryogenesis abundant proteins (LEAPs). This heterogeneous group of anhydrobiosis-related intrinsically disordered proteins forms mostly random conformation when fully hydrated, turning into compact α-helices during desiccation [2]. Based on in vitro studies, LEAPs can be involved in water binding, ion sequestration, stabilization of both membrane and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation.
Here, we identify 164 members of LEA gene family in endemic and relict resurrection species Ramonda serbica by integrating previously done de novo transcriptome and homologues protein motifs. Identified LEAPs were classification into six groups according to Protein family (PFAM) database and the most populated group was LEA4 containing 47% of total identified LEAPs. By using four secondary structure predictors, we showed that this group exhibited a high propensity to form amphipathic α-helices (81% of total sequence length is predicted to form α-helical structure). This implies that charged residues might be exposed to the solvent, while hydrophobic amino acids might interact with lipid bilayers or with other target proteins in the cell. In addition, as predicted by several bioinformatic tools, more than 70% of identified LEAPs were found to be highly disordered (~64%). Structural characterization of LEAPs is a key to understand their function and regulation of their intrinsic structural disorder-to-order transition during desiccation. These findings will promote transformative advancements in various fields, such as the development of new strategies in neurodegenerative disorders, cell preservation technology and the improvement of crop drought tolerance.",
publisher = "Novi Sad : Faculty of Sciences, Department of Biology and Ecology",
journal = "Biologia Serbica",
title = "De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins",
volume = "43",
number = "1 (spec. ed.)",
pages = "65",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4503"
}
Pantelić, A., Stevanović, S., Kilibarda, N.,& Vidović, M.. (2021). De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins. in Biologia Serbica
Novi Sad : Faculty of Sciences, Department of Biology and Ecology., 43(1 (spec. ed.)), 65.
https://hdl.handle.net/21.15107/rcub_cherry_4503
Pantelić A, Stevanović S, Kilibarda N, Vidović M. De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins. in Biologia Serbica. 2021;43(1 (spec. ed.)):65.
https://hdl.handle.net/21.15107/rcub_cherry_4503 .
Pantelić, Ana, Stevanović, Strahinja, Kilibarda, Nataša, Vidović, Marija, "De Novo Transcriptome Sequencing of Ramonda serbica: Identification of Late Embryogenesis Abundant Proteins" in Biologia Serbica, 43, no. 1 (spec. ed.) (2021):65,
https://hdl.handle.net/21.15107/rcub_cherry_4503 .

De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins

Pantelić, Ana; Stevanović, Strahinja; Kilibarda, Nataša; Vidović, Marija

(Belgrade BioInformatics, 2021)

TY  - GEN
AU  - Pantelić, Ana
AU  - Stevanović, Strahinja
AU  - Kilibarda, Nataša
AU  - Vidović, Marija
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4506
PB  - Belgrade BioInformatics
T2  - Belgrade BioInformatics Conference 2021
T1  - De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4506
ER  - 
@misc{
author = "Pantelić, Ana and Stevanović, Strahinja and Kilibarda, Nataša and Vidović, Marija",
year = "2021",
publisher = "Belgrade BioInformatics",
journal = "Belgrade BioInformatics Conference 2021",
title = "De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4506"
}
Pantelić, A., Stevanović, S., Kilibarda, N.,& Vidović, M.. (2021). De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins. in Belgrade BioInformatics Conference 2021
Belgrade BioInformatics..
https://hdl.handle.net/21.15107/rcub_cherry_4506
Pantelić A, Stevanović S, Kilibarda N, Vidović M. De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins. in Belgrade BioInformatics Conference 2021. 2021;.
https://hdl.handle.net/21.15107/rcub_cherry_4506 .
Pantelić, Ana, Stevanović, Strahinja, Kilibarda, Nataša, Vidović, Marija, "De Novo Transcriptome Sequencing of Ramonda serbica : Identification of Late Embryogenesis Abundant Proteins" in Belgrade BioInformatics Conference 2021 (2021),
https://hdl.handle.net/21.15107/rcub_cherry_4506 .

Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli

Pantelić, Ana

(2021)

TY  - THES
AU  - Pantelić, Ana
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4602
AB  - Cilj ove master teze je optimizacija procesa dobijanja prečišćenog proteina zastupljenog u kasnoj fazi embriogeneze (LEAP) iz biljke vaskrsnice Ramonda serbica tehnologijom rekombinantne DNK. Ova procedura obezbeđuje značajne količine LEA proteina radi daljih strukturnih i funkcionalnih analiza, prevashodno za ispitivanje potencijalnih interakcija sa rekombinantnim humanim α-sinukelinom, dobijenog u mom Završnom radu. Detalji vezani za opis tehnika rekombinantne DNK tehnologije i potencijalnu problematiku su opisani u pomenutom Završnom radu.
T1  - Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli
SP  - 2
EP  - 94
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4602
ER  - 
@mastersthesis{
author = "Pantelić, Ana",
year = "2021",
abstract = "Cilj ove master teze je optimizacija procesa dobijanja prečišćenog proteina zastupljenog u kasnoj fazi embriogeneze (LEAP) iz biljke vaskrsnice Ramonda serbica tehnologijom rekombinantne DNK. Ova procedura obezbeđuje značajne količine LEA proteina radi daljih strukturnih i funkcionalnih analiza, prevashodno za ispitivanje potencijalnih interakcija sa rekombinantnim humanim α-sinukelinom, dobijenog u mom Završnom radu. Detalji vezani za opis tehnika rekombinantne DNK tehnologije i potencijalnu problematiku su opisani u pomenutom Završnom radu.",
title = "Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli",
pages = "2-94",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4602"
}
Pantelić, A.. (2021). Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli. , 2-94.
https://hdl.handle.net/21.15107/rcub_cherry_4602
Pantelić A. Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli. 2021;:2-94.
https://hdl.handle.net/21.15107/rcub_cherry_4602 .
Pantelić, Ana, "Optimizacija protokola proizvodnje proteina zastupljenog u kasnoj fazi embriogeneze (RsLEA_30) iz biljke vaskrsnice Ramonda serbica tehnikom rekombinantne DNK u bakteriji Escherichia coli" (2021):2-94,
https://hdl.handle.net/21.15107/rcub_cherry_4602 .

Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach

Milić Komić, Sonja; Stevanović, Strahinja; Vidović, Marija

(2021)

TY  - CONF
AU  - Milić Komić, Sonja
AU  - Stevanović, Strahinja
AU  - Vidović, Marija
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4507
AB  - Ramonda serbica Panc. is a resurrection plant that can survive long desiccation periods (extreme loss of cellular water). The accumulation of late embryogenesis abundant proteins (LEAPs) is a crucial step in desiccation tolerance mechanism. Based on in vitro studies, LEAPs can be involved in antioxidative defense, ion sequestration, structural stabilization of both membranes and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here we investigated the antioxidative potential of LEAPs identified by de novo transcriptomics of R. serbica, based on their primary and secondary confirmation. In our
previous work [1], we displayed the antioxidative capacity of 20 free proteogenic amino acids
(FAA) through determining their hydroxyl radical (•OH, generated in Fenton reaction) scavenging
rate by using electron paramagnetic resonance. These results served as a basis for generating a model for prediction of •OH scavenging activity for selected proteins. In addition, the model was built based on protein primary sequences, hydrophobicity, 3D structure and predicted solvent accessible area. Manually curated data for peptides and proteins with experimentally determined •OH scavenging rate were used for training and testing. The model was fed into machine learning algorithm and •OH scavenging potential scale was created using IC50 values. By applying our model, we classified 164 LEAPs according to their potential for •OH scavenging. Further work will focus on the experimental evaluation of the obtained model by measuring of the rate of • OH scavenging in the presence of recombinantly produced LEAPs.
C3  - Annual Meeting, SFRR-E 2021 Belgrade, Serbia, 15-18 June
T1  - Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4507
ER  - 
@conference{
author = "Milić Komić, Sonja and Stevanović, Strahinja and Vidović, Marija",
year = "2021",
abstract = "Ramonda serbica Panc. is a resurrection plant that can survive long desiccation periods (extreme loss of cellular water). The accumulation of late embryogenesis abundant proteins (LEAPs) is a crucial step in desiccation tolerance mechanism. Based on in vitro studies, LEAPs can be involved in antioxidative defense, ion sequestration, structural stabilization of both membranes and enzymes during freezing or drying, while by forming intracellular proteinaceous condensates they increase structural integrity and intracellular viscosity of cells during desiccation. Here we investigated the antioxidative potential of LEAPs identified by de novo transcriptomics of R. serbica, based on their primary and secondary confirmation. In our
previous work [1], we displayed the antioxidative capacity of 20 free proteogenic amino acids
(FAA) through determining their hydroxyl radical (•OH, generated in Fenton reaction) scavenging
rate by using electron paramagnetic resonance. These results served as a basis for generating a model for prediction of •OH scavenging activity for selected proteins. In addition, the model was built based on protein primary sequences, hydrophobicity, 3D structure and predicted solvent accessible area. Manually curated data for peptides and proteins with experimentally determined •OH scavenging rate were used for training and testing. The model was fed into machine learning algorithm and •OH scavenging potential scale was created using IC50 values. By applying our model, we classified 164 LEAPs according to their potential for •OH scavenging. Further work will focus on the experimental evaluation of the obtained model by measuring of the rate of • OH scavenging in the presence of recombinantly produced LEAPs.",
journal = "Annual Meeting, SFRR-E 2021 Belgrade, Serbia, 15-18 June",
title = "Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4507"
}
Milić Komić, S., Stevanović, S.,& Vidović, M.. (2021). Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach. in Annual Meeting, SFRR-E 2021 Belgrade, Serbia, 15-18 June.
https://hdl.handle.net/21.15107/rcub_cherry_4507
Milić Komić S, Stevanović S, Vidović M. Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach. in Annual Meeting, SFRR-E 2021 Belgrade, Serbia, 15-18 June. 2021;.
https://hdl.handle.net/21.15107/rcub_cherry_4507 .
Milić Komić, Sonja, Stevanović, Strahinja, Vidović, Marija, "Hydroxyl radical scavenging potential of the late embryogenesis abundant proteins (LEA) proteins from Ramonda serbica – in silico approach" in Annual Meeting, SFRR-E 2021 Belgrade, Serbia, 15-18 June (2021),
https://hdl.handle.net/21.15107/rcub_cherry_4507 .

In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship

Stevanović, Strahinja; Vidović, Marija

(2021)

TY  - CONF
AU  - Stevanović, Strahinja
AU  - Vidović, Marija
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4508
AB  - Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterised by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilisation of membranes and enzymes during freezing or drying [2].
Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 164 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using PDB structures as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. 
The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.
C3  - Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5
T1  - In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4508
ER  - 
@conference{
author = "Stevanović, Strahinja and Vidović, Marija",
year = "2021",
abstract = "Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterised by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilisation of membranes and enzymes during freezing or drying [2].
Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 164 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using PDB structures as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. 
The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.",
journal = "Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5",
title = "In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4508"
}
Stevanović, S.,& Vidović, M.. (2021). In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship. in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5.
https://hdl.handle.net/21.15107/rcub_cherry_4508
Stevanović S, Vidović M. In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship. in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5. 2021;.
https://hdl.handle.net/21.15107/rcub_cherry_4508 .
Stevanović, Strahinja, Vidović, Marija, "In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship" in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5 (2021),
https://hdl.handle.net/21.15107/rcub_cherry_4508 .

De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance

Vidović, Marija; Stevanović, Strahinja; Veljović-Jovanović, Sonja

(2021)

TY  - CONF
AU  - Vidović, Marija
AU  - Stevanović, Strahinja
AU  - Veljović-Jovanović, Sonja
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4509
AB  - Ramonda serbica Panc. is a resurrection plant that can survive a long period of severe dehydration-desiccation. Desiccation induces cellular membrane integrity loss, protein aggregation, and denaturation, as well as accelerated generation of reactive oxygen species. However, R. serbica can fully recover its metabolic functions already one day upon watering [1]. The aim of our study was to obtain more insight into the desiccation tolerance mechanisms by differential de novo transcriptomics of hydrated (HL) and desiccated leaves (DL). 
For R. serbica transcriptome construction, the total high-quality RNA from HL and DL was extracted according to our previously optimised protocol [2]. Highly purified cDNA libraries were sequenced on an Illumina Hi-Seq platform. The ambiguous nucleotides, adapter sequences, and low-quality sequences were trimmed, and the quality of the reads was checked before and after the trimming. In total, 39608813 (with Q30=94%) and 37482969 (with Q30=94.1%) clean reads were obtained in HL and DL, respectively, and used to perform transcriptome assembly by Trinity software. After removing the redundancy, 189456 transcripts with 189003 unigenes were obtained (32.6% with the length between 500-1kbp).
Comparative analysis revealed that a large portion of R. serbica sequences (49.1%) was similar to sequences found in the genome of another resurrection plant Boea hygrometrica. Furthermore, among obtained unigenes, 64.6% and 42.3% were annotated by NCBI non-redundant protein and nucleotide sequences database (db), 23% by PFAM db, 22.5% by Clusters of Orthologous Groups of proteins db, 48.02% by Swiss-Prot db, 23 % KEGG db and 13.73 by Gene Ontology db. The majority of annotated genes were associated with translation, ribosomal structure, posttranslational modifications, protein turnover, signalling pathways and cytoskeleton and encoded chaperonins and late embryogenesis abundant (LEA) proteins. 
Aiming to provide a list of candidates involved in the desiccation tolerance in R. serbica we analysed differentially expressed genes in HL and DL. Genes associated with transmembrane transport, reproduction, cell proliferation, and protein folding were up-regulated in HL compared with DL. On the other hand, genes encoding proteins involved in cell wall architecture, LEA proteins and antioxidative defence were up-regulated in DL.
C3  - Biologia Serbica
T1  - De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance
VL  - 43
IS  - 1 (spec. ed.)
SP  - 75
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4509
ER  - 
@conference{
author = "Vidović, Marija and Stevanović, Strahinja and Veljović-Jovanović, Sonja",
year = "2021",
abstract = "Ramonda serbica Panc. is a resurrection plant that can survive a long period of severe dehydration-desiccation. Desiccation induces cellular membrane integrity loss, protein aggregation, and denaturation, as well as accelerated generation of reactive oxygen species. However, R. serbica can fully recover its metabolic functions already one day upon watering [1]. The aim of our study was to obtain more insight into the desiccation tolerance mechanisms by differential de novo transcriptomics of hydrated (HL) and desiccated leaves (DL). 
For R. serbica transcriptome construction, the total high-quality RNA from HL and DL was extracted according to our previously optimised protocol [2]. Highly purified cDNA libraries were sequenced on an Illumina Hi-Seq platform. The ambiguous nucleotides, adapter sequences, and low-quality sequences were trimmed, and the quality of the reads was checked before and after the trimming. In total, 39608813 (with Q30=94%) and 37482969 (with Q30=94.1%) clean reads were obtained in HL and DL, respectively, and used to perform transcriptome assembly by Trinity software. After removing the redundancy, 189456 transcripts with 189003 unigenes were obtained (32.6% with the length between 500-1kbp).
Comparative analysis revealed that a large portion of R. serbica sequences (49.1%) was similar to sequences found in the genome of another resurrection plant Boea hygrometrica. Furthermore, among obtained unigenes, 64.6% and 42.3% were annotated by NCBI non-redundant protein and nucleotide sequences database (db), 23% by PFAM db, 22.5% by Clusters of Orthologous Groups of proteins db, 48.02% by Swiss-Prot db, 23 % KEGG db and 13.73 by Gene Ontology db. The majority of annotated genes were associated with translation, ribosomal structure, posttranslational modifications, protein turnover, signalling pathways and cytoskeleton and encoded chaperonins and late embryogenesis abundant (LEA) proteins. 
Aiming to provide a list of candidates involved in the desiccation tolerance in R. serbica we analysed differentially expressed genes in HL and DL. Genes associated with transmembrane transport, reproduction, cell proliferation, and protein folding were up-regulated in HL compared with DL. On the other hand, genes encoding proteins involved in cell wall architecture, LEA proteins and antioxidative defence were up-regulated in DL.",
journal = "Biologia Serbica",
title = "De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance",
volume = "43",
number = "1 (spec. ed.)",
pages = "75",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4509"
}
Vidović, M., Stevanović, S.,& Veljović-Jovanović, S.. (2021). De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance. in Biologia Serbica, 43(1 (spec. ed.)), 75.
https://hdl.handle.net/21.15107/rcub_cherry_4509
Vidović M, Stevanović S, Veljović-Jovanović S. De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance. in Biologia Serbica. 2021;43(1 (spec. ed.)):75.
https://hdl.handle.net/21.15107/rcub_cherry_4509 .
Vidović, Marija, Stevanović, Strahinja, Veljović-Jovanović, Sonja, "De Novo Transcriptome Sequencing of Ramonda serbica: Identification of the Candidate Genes Involved in the Desiccation Tolerance" in Biologia Serbica, 43, no. 1 (spec. ed.) (2021):75,
https://hdl.handle.net/21.15107/rcub_cherry_4509 .

Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK

Marković, Nemanja

(2021)

TY  - THES
AU  - Marković, Nemanja
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4598
AB  - Cilj ovog master rada je optimizacija procesa dobijanja prečišćenog LEA proteina (AtLEA25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNA. Ovom procedurom je potrebno dobiti značajne količine LEA proteina radi daljih strukturnih i funkcionalnih analiza, prevashodno za ispitivanje potencijalnih interakcija sa rekombinantnim humanim α – sinukelinom.
T1  - Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK
SP  - 2
EP  - 82
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4598
ER  - 
@mastersthesis{
author = "Marković, Nemanja",
year = "2021",
abstract = "Cilj ovog master rada je optimizacija procesa dobijanja prečišćenog LEA proteina (AtLEA25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNA. Ovom procedurom je potrebno dobiti značajne količine LEA proteina radi daljih strukturnih i funkcionalnih analiza, prevashodno za ispitivanje potencijalnih interakcija sa rekombinantnim humanim α – sinukelinom.",
title = "Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK",
pages = "2-82",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4598"
}
Marković, N.. (2021). Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK. , 2-82.
https://hdl.handle.net/21.15107/rcub_cherry_4598
Marković N. Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK. 2021;:2-82.
https://hdl.handle.net/21.15107/rcub_cherry_4598 .
Marković, Nemanja, "Optimizacija protokola za efikasnu proizvodnju proteina zastupljenog u kasnoj fazi embriogeneze (AtLEA_25) iz biljke Arabidopsis thaliana tehnologijom rekombinantne DNK" (2021):2-82,
https://hdl.handle.net/21.15107/rcub_cherry_4598 .

In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship

Stevanović, Strahinja; Vidović, Marija

(2021)

TY  - CONF
AU  - Stevanović, Strahinja
AU  - Vidović, Marija
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4649
AB  - Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterized by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilization of membranes and enzymes during freezing or drying [2]. Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 153 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using Protein Data Bank structure information as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.
C3  - Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5
T1  - In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4649
ER  - 
@conference{
author = "Stevanović, Strahinja and Vidović, Marija",
year = "2021",
abstract = "Desiccation or extreme water loss leads to protein denaturation, aggregation, and degradation and impairs membrane lipid fluidity, resulting in loss of membrane integrity at the cellular level. The induction of late embryogenesis abundant proteins (LEAPs) is considered an essential component of desiccation tolerance strategy in so-called resurrection plants. This heterogeneous group of hydrophilic, non-globular proteins is characterized by a high structural plasticity that allows them to adopt a random conformation in aqueous solutions that transforms into α-helices during dehydration [1]. Therefore, LEAPs can interact with various ligands and partners, including ion sequestration and stabilization of membranes and enzymes during freezing or drying [2]. Our new transcriptome database of an endemic resurrection species Ramonda serbica allowed us to identify 153 members of the LEA gene family. LEAPs of this sample data have an average primary sequence similarity and identity of 10% and 6%, respectively, but with a high variance (141 and 108), which means that the sample proteins can be classified based on domain homology. The averaging is based on multiple sequence alignment and the variance is estimated using pairwise sequence alignment scores. Accordingly, all identified LEAPs were clustered into six groups based on protein families (PFAM). Among these groups, LEAPs differ significantly in their secondary structure, disorder propensity and aggregation potential. Furthermore, we built homology models using Protein Data Bank structure information as templates. For each group, an ensemble of superimposed 3D homology models was analyzed. The information obtained from the representative structural models is key to understanding the function of LEAPs and the regulation of their intrinsic structural disorder-to-order transition during desiccation. This will pave the way for the identification of LEAPs endogenous partners and their targets in the cell and provide further insights into the protective mechanisms of desiccation tolerance.",
journal = "Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5",
title = "In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4649"
}
Stevanović, S.,& Vidović, M.. (2021). In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship. in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5.
https://hdl.handle.net/21.15107/rcub_cherry_4649
Stevanović S, Vidović M. In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship. in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5. 2021;.
https://hdl.handle.net/21.15107/rcub_cherry_4649 .
Stevanović, Strahinja, Vidović, Marija, "In silico structural survey of newly identified late embryogenesis abundant proteins (LEAPs) from Ramonda serbica and their structure - function relationship" in Virtual symposium celebrating the 50th anniversary of the Protein Data Bank, May 4–5 (2021),
https://hdl.handle.net/21.15107/rcub_cherry_4649 .

Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli

Savić, Aleksa D.

(2021)

TY  - THES
AU  - Savić, Aleksa D.
PY  - 2021
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4736
AB  - Humani α-sinuklein je protein zastupljen u završecima presinaptičkih neurona. Agregati ovog proteina su pronađeni u okviru amiloidnih fibrila i Lewy-jevih tela u mozgovima pacijenata sa Alchajmerovom, Parkinsonovom i drugim bolestima i smatra se da su usko povezani sa etiologijom ovih bolesti. Da bi se ispitale priroda ovog proteina i njegove oligomerizacije in vitro potrebne su velike količine prečišćenog proteina. U ovom radu opisana je konstrukcija vektora za ekspresiju humanog  α-sinukleina fluorescentno obeleženog mCerulean3 proteinom sa His6, odnosno His8 tagom i uvedenim proteolitičkim mestom za TEV proteazu, koji nakon digestije oslobađa  α-sinuklein sa nativom N-terminalnom aminokiselinom, a polazeći od komercijalno dostupnog pDUET-A- α-sinuclein-mCerulean3 vektora. Za dobijanje ekspresionog konstrukta, prvo je sintetisan insert PCR-om iz više ciklusa, nakon čega su PCR proizvodi subklonirani u pET-20b vektor, a zatim prebačeni u pDUET vektor. Sekvenca inserta u dobijenim konstruktima je potvrđena sekvenciranjem sa opdgovarajućim prajmerima.
T1  - Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli
SP  - 1
EP  - 69
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4736
ER  - 
@misc{
author = "Savić, Aleksa D.",
year = "2021",
abstract = "Humani α-sinuklein je protein zastupljen u završecima presinaptičkih neurona. Agregati ovog proteina su pronađeni u okviru amiloidnih fibrila i Lewy-jevih tela u mozgovima pacijenata sa Alchajmerovom, Parkinsonovom i drugim bolestima i smatra se da su usko povezani sa etiologijom ovih bolesti. Da bi se ispitale priroda ovog proteina i njegove oligomerizacije in vitro potrebne su velike količine prečišćenog proteina. U ovom radu opisana je konstrukcija vektora za ekspresiju humanog  α-sinukleina fluorescentno obeleženog mCerulean3 proteinom sa His6, odnosno His8 tagom i uvedenim proteolitičkim mestom za TEV proteazu, koji nakon digestije oslobađa  α-sinuklein sa nativom N-terminalnom aminokiselinom, a polazeći od komercijalno dostupnog pDUET-A- α-sinuclein-mCerulean3 vektora. Za dobijanje ekspresionog konstrukta, prvo je sintetisan insert PCR-om iz više ciklusa, nakon čega su PCR proizvodi subklonirani u pET-20b vektor, a zatim prebačeni u pDUET vektor. Sekvenca inserta u dobijenim konstruktima je potvrđena sekvenciranjem sa opdgovarajućim prajmerima.",
title = "Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli",
pages = "1-69",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4736"
}
Savić, A. D.. (2021). Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli. , 1-69.
https://hdl.handle.net/21.15107/rcub_cherry_4736
Savić AD. Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli. 2021;:1-69.
https://hdl.handle.net/21.15107/rcub_cherry_4736 .
Savić, Aleksa D., "Kloniranje fluorescentno obeleženog humanog α-sinukleina u vektore za ekspresiju u bakteriji Escherichia coli" (2021):1-69,
https://hdl.handle.net/21.15107/rcub_cherry_4736 .

Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2.

Vidović, Marija; Franchin, Cinzia; Morina, Filis; Veljović-Jovanović, Sonja; Masi, Antonio; Arrigoni, Giorgio

(SpringerLink, 2020)

TY  - DATA
AU  - Vidović, Marija
AU  - Franchin, Cinzia
AU  - Morina, Filis
AU  - Veljović-Jovanović, Sonja
AU  - Masi, Antonio
AU  - Arrigoni, Giorgio
PY  - 2020
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4504
PB  - SpringerLink
T2  - Analytical and Bioanalytical Chemistry
T1  - Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2.
UR  - https://hdl.handle.net/21.15107/rcub_cherry_4504
ER  - 
@misc{
author = "Vidović, Marija and Franchin, Cinzia and Morina, Filis and Veljović-Jovanović, Sonja and Masi, Antonio and Arrigoni, Giorgio",
year = "2020",
publisher = "SpringerLink",
journal = "Analytical and Bioanalytical Chemistry",
title = "Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2.",
url = "https://hdl.handle.net/21.15107/rcub_cherry_4504"
}
Vidović, M., Franchin, C., Morina, F., Veljović-Jovanović, S., Masi, A.,& Arrigoni, G.. (2020). Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2.. in Analytical and Bioanalytical Chemistry
SpringerLink..
https://hdl.handle.net/21.15107/rcub_cherry_4504
Vidović M, Franchin C, Morina F, Veljović-Jovanović S, Masi A, Arrigoni G. Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2.. in Analytical and Bioanalytical Chemistry. 2020;.
https://hdl.handle.net/21.15107/rcub_cherry_4504 .
Vidović, Marija, Franchin, Cinzia, Morina, Filis, Veljović-Jovanović, Sonja, Masi, Antonio, Arrigoni, Giorgio, "Supplementary data for the article: Vidović, M.; Franchin, C.; Morina, F.; Veljović-Jovanović, S.; Masi, A.; Arrigoni, G. Efficient Protein Extraction for Shotgun Proteomics from Hydrated and Desiccated Leaves of Resurrection Ramonda Serbica Plants. Anal Bioanal Chem 2020, 412 (30), 8299–8312. https://doi.org/10.1007/s00216-020-02965-2." in Analytical and Bioanalytical Chemistry (2020),
https://hdl.handle.net/21.15107/rcub_cherry_4504 .

Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants

Vidović, Marija; Franchin, Cinzia; Morina, Filis; Veljović-Jovanović, Sonja; Masi, Antonio; Arrigoni, Giorgio

(2020)

TY  - JOUR
AU  - Vidović, Marija
AU  - Franchin, Cinzia
AU  - Morina, Filis
AU  - Veljović-Jovanović, Sonja
AU  - Masi, Antonio
AU  - Arrigoni, Giorgio
PY  - 2020
UR  - http://www.ncbi.nlm.nih.gov/pubmed/33037906
UR  - https://cherry.chem.bg.ac.rs/handle/123456789/4505
AB  - Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.
T2  - Analytical and Bioanalytical Chemistry
T1  - Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants
VL  - 412
IS  - 30
SP  - 8299
EP  - 8312
DO  - 10.1007/s00216-020-02965-2
ER  - 
@article{
author = "Vidović, Marija and Franchin, Cinzia and Morina, Filis and Veljović-Jovanović, Sonja and Masi, Antonio and Arrigoni, Giorgio",
year = "2020",
abstract = "Resurrection plant Ramonda serbica is a suitable model to investigate vegetative desiccation tolerance. However, the detailed study of these mechanisms at the protein level is hampered by the severe tissue water loss, high amount of phenolics and polysaccharide, and possible protein modifications and aggregations during the extraction and purification steps. When applied to R. serbica leaves, widely used protein extraction protocols containing polyvinylpolypyrrolidone and ascorbate, as well as the phenol/SDS/buffer-based protocol recommended for recalcitrant plant tissues failed to eliminate persistent contamination and ensure high protein quality. Here we compared three protein extraction approaches aiming to establish the optimal one for both hydrated and desiccated R. serbica leaves. To evaluate the efficacy of these protocols by shotgun proteomics, we also created the first R. serbica annotated transcriptome database, available at http://www.biomed.unipd.it/filearrigoni/Trinity_Sample_RT2.fasta . The detergent-free phenol-based extraction combined with dodecyl-β-D-maltoside-assisted extraction enabled high-yield and high-purity protein extracts. The phenol-based protocol improved the protein-band resolution, band number, and intensity upon electrophoresis, and increased the protein yield and the number of identified peptides and protein groups by LC-MS/MS. Additionally, dodecyl-β-D-maltoside enabled solubilisation and identification of more membrane-associated proteins. The presented study paves the way for investigating the desiccation tolerance in R. serbica, and we recommend this protocol for similar recalcitrant plant material.",
journal = "Analytical and Bioanalytical Chemistry",
title = "Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants",
volume = "412",
number = "30",
pages = "8299-8312",
doi = "10.1007/s00216-020-02965-2"
}
Vidović, M., Franchin, C., Morina, F., Veljović-Jovanović, S., Masi, A.,& Arrigoni, G.. (2020). Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. in Analytical and Bioanalytical Chemistry, 412(30), 8299-8312.
https://doi.org/10.1007/s00216-020-02965-2
Vidović M, Franchin C, Morina F, Veljović-Jovanović S, Masi A, Arrigoni G. Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants. in Analytical and Bioanalytical Chemistry. 2020;412(30):8299-8312.
doi:10.1007/s00216-020-02965-2 .
Vidović, Marija, Franchin, Cinzia, Morina, Filis, Veljović-Jovanović, Sonja, Masi, Antonio, Arrigoni, Giorgio, "Efficient protein extraction for shotgun proteomics from hydrated and desiccated leaves of resurrection Ramonda serbica plants" in Analytical and Bioanalytical Chemistry, 412, no. 30 (2020):8299-8312,
https://doi.org/10.1007/s00216-020-02965-2 . .
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