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Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli
Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli
dc.creator | Savić, Aleksa D. | |
dc.creator | Vidović, Marija | |
dc.creator | Radosavljević, Jelena | |
dc.date.accessioned | 2022-06-30T13:19:05Z | |
dc.date.available | 2022-06-30T13:19:05Z | |
dc.date.issued | 2022 | |
dc.identifier.isbn | 978-86-7132-079-5 | |
dc.identifier.uri | http://cherry.chem.bg.ac.rs/handle/123456789/5374 | |
dc.description.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. | sr |
dc.description.abstract | 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. | en |
dc.language.iso | sr | sr |
dc.language.iso | en | sr |
dc.publisher | Beograd : Srpsko hemijsko društvo | sr |
dc.relation | info:eu-repo/grantAgreement/ScienceFundRS/Promis/6039663/RS// | sr |
dc.relation | info:eu-repo/grantAgreement/MESTD/inst-2020/200168/RS// | |
dc.rights | openAccess | sr |
dc.rights.uri | https://creativecommons.org/licenses/by/4.0/ | |
dc.source | 58. Savetovanje Srpskog hemijskog društva, Kratki izvodi radova, Beograd 9. i 10. jun 2022. godine | sr |
dc.title | Kloniranje i ekspresija fluorescentno obeleženog )-sinukleina u bakteriji Escherichia coli | sr |
dc.title | Cloning and expression of fluorescently labeled )-synuclein in Eschierichia coli | sr |
dc.type | conferenceObject | sr |
dc.rights.license | BY | sr |
dc.description.other | Abstract: [https://cherry.chem.bg.ac.rs/handle/123456789/5373] | |
dc.type.version | publishedVersion | sr |
dc.identifier.fulltext | http://cherry.chem.bg.ac.rs/bitstream/id/30570/SHD_PROMIS_Savic-12.pdf | |
dc.identifier.rcub | https://hdl.handle.net/21.15107/rcub_cherry_5374 |