članak: 1 od 1  
Journal of the Serbian Chemical Society
2009, vol. 74, br. 6, str. 607-618
jezik rada: engleski
izvorni naučni članak
doi:10.2298/JSC0906607M

Uspešna proizvodnja rekombinantne aspartatne proteinaze heljde bogate cisteinom u E.coli
Institut za molekularnu genetiku i genetičko inženjerstvo, Beograd

e-adresa: heljda@sezampro.rs

Projekat Ministarstva nauke Republike Srbije, br. 143017

Sažetak

U ovom radu predstavljena je ekspresija rekombinantne atipične aspartatne proteinaze heljde (Fagopyrum esculentum) bogate cisteinom, gde su testirana različita ekspresiona svojstva pet sojeva E. coli. Takođe je analiziran i uticaj fuzionih partnera (His6 i MBP) na efikasnost ekspresije. U slučaju His6-FeAPL1, dobijena je velika količina nerastvornog proteina, smeštenog u inkluzionim telima. S druge strane, MBP-FeAPL1 je bio lokalizovan i u citoplazmi i u inkluzionim telima u oba upotrebljena soja E. coli (BL21 i Rosetta-gami). Međutim, samo za rekombinantni protein proizveden u soju Rosetta-gami, dokazana je proteolitička aktivnost na supstratu BSA, pri pH 3,0. Rezultati su takođe ukazali da FeAPL1 sadrži PRO segment, čije je odstranjivanje neophodno za njegovu proteolitičku aktivnost. Aktivnost FeAPL1, pokazana samo u soju Rosetta-gami, gde je moguće formiranje disulfidnih veza, ukazuje na značaj 12 cisteina u uspostavljanju pravilne strukture koja omogućava funkcionalnost enzima.

Ključne reči

aspartic protease; His tag; inclusion bodies; MBP; recombinant protein

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