Prikaz osnovnih podataka o dokumentu
Optimization of the heterologous expression of banana glucanase in Escherichia coli
Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli
dc.creator | Abughren, Mohamed | |
dc.creator | Popović, Milica | |
dc.creator | Dimitrijević, Rajna | |
dc.creator | Burazer, Lidija | |
dc.creator | Grozdanović, Milica | |
dc.creator | Atanasković-Marković, Marina | |
dc.creator | Gavrović-Jankulović, Marija | |
dc.date.accessioned | 2021-02-18T10:38:44Z | |
dc.date.available | 2021-02-18T10:38:44Z | |
dc.date.issued | 2012 | |
dc.identifier.issn | 0352-5139 | |
dc.identifier.uri | http://intor.torlakinstitut.com/handle/123456789/350 | |
dc.description.abstract | For the heterologous production of a banana glucanase in Escherichia coli, its gene (GenBank GQ268963) was cloned into a pG EX-4T expression vector as a fusion protein with glutathione-S-transferase (GST). BL21 cells transformed with the GST-Mus a 5 con struct were employed for production of the protein induced by 1 mM isopropyl-β-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°C upon addition of IPTG. Recombinant GST-Mus a 5 purified by glutathione affinity chromatography revealed a molecular mass of a bout 60 kDa. The IgE and IgG reactivity of the rGST-Mus a 5 was confirmed by dot blot an 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.abstract | Za 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.publisher | Srpsko hemijsko društvo, Beograd | |
dc.relation | info:eu-repo/grantAgreement/MESTD/Basic Research (BR or ON)/172049/RS// | |
dc.relation.isreferencedby | https://hdl.handle.net/21.15107/rcub_intor_364 | |
dc.rights | openAccess | |
dc.rights.uri | https://creativecommons.org/licenses/by-nc-nd/4.0/ | |
dc.source | Journal of the Serbian Chemical Society | |
dc.subject | food allergen | en |
dc.subject | protein expression | en |
dc.subject | glucanase | en |
dc.title | Optimization of the heterologous expression of banana glucanase in Escherichia coli | en |
dc.title | Optimizacija heterologe proizvodnje glukanaze iz banane u E. coli | sr |
dc.type | article | |
dc.rights.license | BY-NC-ND | |
dc.citation.epage | 52 | |
dc.citation.issue | 1 | |
dc.citation.other | 77(1): 43-52 | |
dc.citation.rank | M23 | |
dc.citation.spage | 43 | |
dc.citation.volume | 77 | |
dc.description.other | Link to the corrected article: [https://hdl.handle.net/21.15107/rcub_intor_364] | |
dc.identifier.doi | 10.2298/JSC110309158A | |
dc.identifier.fulltext | http://intor.torlakinstitut.com/bitstream/id/193/347.pdf | |
dc.identifier.scopus | 2-s2.0-84858634561 | |
dc.identifier.wos | 000300386100005 | |
dc.type.version | publishedVersion |