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Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli

dc.creatorAbughren, Mohamed
dc.creatorPopović, Milica
dc.creatorDimitrijevic, Rajna
dc.creatorBurazer, Lidija M.
dc.creatorGrozdanović, Milica
dc.creatorAtanasković-Marković, Marina
dc.creatorGavrović-Jankulović, Marija
dc.date.accessioned2018-11-22T00:20:27Z
dc.date.available2018-11-22T00:20:27Z
dc.date.issued2012
dc.identifier.issn0352-5139
dc.identifier.urihttp://cherry.chem.bg.ac.rs/handle/123456789/1246
dc.description.abstractFor the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pGEX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 construct were employed for production of the protein induced by 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG). The conditions for protein expression were optimized by varying the temperature (25, 30 and 37 C) and duration of protein expression (3, 6 and 12 h). The level of protein production was analyzed by densitometry of the sodium dodecyl sulfate-polyacrylamide gel (SDS-PAG) after electrophoretic resolution of the respective cell lysates. The optimal protein expression for downstream processing was obtained after 12 h of cell growth at 25 degrees C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of about 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot analysis with sera of individual patients from subjects with banana allergy and polyclonal rabbit antibodies against banana extract, respectively. The purified recombinant glucanase is a potential candidate for banana allergy diagnosis.en
dc.description.abstractZa potrebe proizvodnje u Escherichia coli gen glukanaze iz banane (GenBank GQ268963) je ukloniran u ekspresioni vektor pGEX-4T sa glutation-S-transferazom (GST). Proizvodnja ovog proteina u ćelijama je indukovana 1 mM izopropil-β-D-tiogalaktopiranozidom (IPTG). Uslovi za ekspresiju proteina su optimizovani variranjem temperature (25, 30 i 37°C) i dužine trajanja proteinske sinteze (3, 6 i 12 h). Nivo proizvodnje proteina je analiziran denzitometrijom SDS-PA gela nakon elektroforetskog razdvajanja ćelijskih lizata. Optimalna proizvodnja proteina za njegovo dalje procesovanje je dobijena gajenjem ćelija nakon dodatka IPTG na 25°C tokom 12 h. Rekombinantni GST-Mus a 5 prečišćen afinitetnom hromatografijom sa glutationom pokazuje molekulsku masu od 60 kDa. IgE i IgG reaktivnost izolovane glukanaze potvrđena je u 'dot blot' sa pojedinačnim serumima osoba alergičnih na bananu, i sa poliklonskim zečijim antitelima na ekstrakt banane, redom. Prečišćena rekombinantna glukanaza je potencijalan kandidat za dijagnozu alergije na bananu.sr
dc.publisherSerbian Chemical Soc, Belgrade
dc.relationinfo:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS//
dc.rightsopenAccess
dc.sourceJournal of the Serbian Chemical Society
dc.subjectfood allergenen
dc.subjectprotein expressionen
dc.subjectprotein expressionen
dc.subjectglucanaseen
dc.subjectglucanaseen
dc.titleOptimization of the heterologous expression of banana glucanase in Escherichia colien
dc.titleOptimizacija heterologe proizvodnje glukanaze iz banane u E. colisr
dc.typearticle
dc.rights.licenseBY-NC-ND
dcterms.abstractДимитријевиц, Рајна; Буразер, Лидија; Гроздановиц, Милица; Aбугхрен, Мохамед; Aтанасковиц-Марковиц, Марина; Гавровић-Јанкуловић, Марија; Поповић, Милица; Оптимизација хетерологе производње глуканазе из банане у Е. цоли; Оптимизација хетерологе производње глуканазе из банане у Е. цоли;
dc.citation.volume77
dc.citation.issue1
dc.citation.spage43
dc.citation.epage52
dc.identifier.wos000300386100005
dc.identifier.doi10.2298/JSC110309158A
dc.citation.other77(1): 43-52
dc.citation.rankM23
dc.type.versionpublishedVersion
dc.identifier.scopus2-s2.0-84858634561
dc.identifier.fulltexthttp://cherry.chem.bg.ac.rs/bitstream/id/8420/1244.pdf
dc.identifier.rcubKon_2268


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