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

Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli

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2022
SHD_PROMIS_Savic-12.pdf (1.060Mb)
Authors
Savić, Aleksa D.
Vidović, Marija S.
Radosavljević, Jelena
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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 successf...ully proteolyzed by TEV protease.

Source:
58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine, 2022, 68-68
Publisher:
  • Beograd : Srpsko hemijsko društvo
Funding / projects:
  • LEAPSyn-SCI - Late Embryogenesis Abundant Proteins: Structural Characterisation and Interaction With Α-Synuclein (RS-6039663)
  • Ministry of Education, Science and Technological Development, Republic of Serbia, Grant no. 200168 (University of Belgrade, Faculty of Chemistry) (RS-200168)
Note:
  • Abstract: https://cherry.chem.bg.ac.rs/handle/123456789/5373

ISBN: 978-86-7132-079-5

[ Google Scholar ]
Handle
https://hdl.handle.net/21.15107/rcub_cherry_5374
URI
http://cherry.chem.bg.ac.rs/handle/123456789/5374
Collections
  • Publikacije
Institution/Community
Hemijski fakultet
TY  - CONF
AU  - Savić, Aleksa D.
AU  - Vidović, Marija S.
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 S. 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. S.,& 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 MS, 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 S., 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 .

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