20.500.12556/RUL-149139 Vzpostavitev modelnega sistema za proizvodnjo nanoteles v kvasovki Saccharomyces cerevisiae Creating a model system for production of nanobodies in yeast Saccharomyces cerevisiae Enodomenska protitelesa (nanotelesa) so v zadnjem desetletju vse bolj priljubljena, zahvaljujoč stabilnosti in enostavni strukturi. Ker sestojé iz zgolj ene polipeptidne verige, jih je možno pravilno zvite pripraviti v bakterijah ali kvasovkah, zato lahko ustvarimo produkcijske sisteme za proizvodnjo, ki so relativno poceni in dostopni. Kvasovka Saccharomyces cerevisiae sodi v skupino varnih organizmov (GRAS), zato so njihovi proteinski produkti široko uporabni – tudi v farmacevtiki. Kvasovke smo transformirali s plazmidom, ki je v vključku vseboval z zapisom za signalni peptid a faktorja spojen zapis za nanotelo proti proteinu bodice virusa SARS CoV 2, inducirali izražanje pod nadzorom inducibilnega promotorja, nato pa iz gojišča izolirali nanotelo. Protein se je uspešno izražal in izločal v gojišče, kar smo ovrednotili s prenosom western. V tej diplomski nalogi smo dokazali, da je predlagani modelni sistem primeren za proizvodnjo izbranega nanotelesa. Single-domain antibodies (nanobodies) have gained popularity in the last decade due to their stability and simple structure. Consisting of only one polypeptide chain, they can be synthesized by bacteria or yeasts as recombinant proteins, and still fold correctly. This allows us to create production systems which are relatively cheap and accessible. Yeast Saccharomyces cerevisiae belongs to GRAS organisms, thus allowing their protein products to have a broad range of applications – including in pharmacy. Upon transforming yeast cells with a plasmid containing a gene for a nanobody against the SARS CoV 2 spike protein following a gene for the alpha-factor leader peptide, the production under the control of an inducible promoter was induced, then the nanobody was isolated. Success of protein expression and secretion into the medium was evaluated using Western blot. In this research, we show that the suggested model system is applicable for production of the chosen nanobody. nanotelo Saccharomyces cerevisiae sekrecija nanobody Saccharomyces cerevisiae secretion true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2023-09-04 12:15:15 2023-09-04 12:15:18 2023-10-06 09:01:04 0000-00-00 00:00:00 2023 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 22811 165400835 Znidar_Zan_-_Vzpostavitev_modelnega_sistema_za_proizvodnjo_nanoteles_v_kvasovki_Saccharomyces_ce.pdf Znidar_Zan_-_Vzpostavitev_modelnega_sistema_za_proizvodnjo_nanoteles_v_kvasovki_Saccharomyces_ce.pdf 1 E35FB7078BF0F53A89ED5CCE9F5F5582 2c52d05f761b46c33ed15c7fa4fd8ff5217af3dc4edbb1c1d18f74152a2cba24 efadb0c9-4b0b-11ee-af86-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=173531 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0