20.500.12556/RUL-190646 Heterologna amilaza kot označevalec za semikvantitativno merjenje izločanja proteinov iz celic Saccharomyces cerevisiae. Heterologous amylase as a marker for semi-quantitative measurement of protein secretion from Saccharomyces cerevisiae cells. Kvasovka Saccharomyces cerevisiae se kot produkcijski organizem za rekombinantne proteine uporablja že vrsto let. Ena izmed pomembnejših lastnosti organizmov, ki se uporabljajo za proizvodnjo proteinov, je sposobnost njihovega izločanja, saj to olajša proces pridobivanja proizvedenih proteinov. V magistrski nalogi smo želeli razviti visokozmogljivo in semikvantitativno metodo, s katero bi lahko spremljali sposobnost izločanja proteinov iz celic S. cerevisiae. Sprva smo morali izbrati primeren encim, s pomočjo katerega bi lahko na trdnih agarskih gojiščih spremljali izločanje modelnega proteina, ter sposobnost izločanja semikvantitativno ovrednotili na podlagi določanja aktivnosti tega encima. Kot najprimernejši encim se je izkazala amilaza, katere aktivnost lahko na gojiščih z dodatkom škroba spremljamo na podlagi nastanka cone zbistritve po izpostavitvi jodovim param. V nadaljevanju smo s pomočjo PCR sestavili gensko kaseto, ki je vsebovala heterologno amilazo in jo s homologno rekombinacijo vstavili v genom kvasovke na mesto gena LYP1. Sledilo je križanje seva, ki je kaseto vseboval, s sevom, ki kasete ni vseboval. Pridobljene segregante smo presejali glede na amilazno aktivnost ter jih v končni fazi eksperimenta med seboj primerjali z namenom določitve segregante z največjo sposobnostjo izločanja heterolognih proteinov. Razvita metoda se je izkazala za obetavno, vendar so za njeno nadaljnjo optimizacijo potrebne dodatne raziskave in poskusi, s katerimi bi lahko potrdili praktično uporabnost pridobljenih rezultatov. The yeast Saccharomyces cerevisiae has been used as a production organism for recombinant proteins for many years. One of the most important characteristics of organisms used for protein production is their secretion capacity, as secretion facilitates the recovery of produced proteins. In this master’s thesis, we aimed to develop a high-throughput, semi-quantitative method for assessing the protein secretion capacity of S. cerevisiae cells. First, a suitable enzyme had to be selected as a model that would enable protein secretion to be monitored on solid agar media and secretion capacity to be semi-quantitatively evaluated based on the enzyme activity. An amylase proved to be the most suitable enzyme, as its activity can be monitored on starch-containing media by the formation of a clearance zone following exposure to iodine vapour. Subsequently, a gene cassette, containing the selected amylase was assembled by PCR and integrated into the yeast genome at the LYP1 locus by homologous recombination. This was followed by mating of a strain which contained the assembled gene cassette, with a strain, which did not contain the gene cassette. The resulting segregants were screened for amylase activity and, in the final stage of the experiment, compared to identify the segregant with the highest capacity for heterologous protein secretion. The developed method showed promising results; however, further research and experiments are required to optimize the method and confirm the applicability of the obtained results. kvasovka izločanje amilaza cona zbistritve križanje yeast secretion amylase clearance zone mating true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-10-31 07:16:47 2026-10-31 07:16:52 2026-11-06 13:11:52 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 302951 304250115 64932.pdf 64932.pdf 1 903E7206F2DEEFE2D3A6DF1D4871283F 98083caa43e464a9bc83784952a24a332dbaff032ac51f28a210b2a91f89d0c8 a14a1333-d4f2-11f1-aea2-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=252204 Biotehniška fakulteta 0 0 0