20.500.12556/RUL-180889 Vrednotenje porotvorne aktivnosti rekombinantnega proteinskega para beauveriolizina A in beauveriolizina B iz glive Beauveria bassiana Determination of pore-forming activity of the recombinant protein pair beauveriolisyin A and beauveriolisyin B from the fungus Beauveria bassiana V sklopu raziskave smo želeli pridobiti topni rekombinantni protein beauveriolzin B (BlyB) v bakteriji Escherichia coli. Poleg tega smo želeli preveriti, ali proteinski par beauveriolzin A (BlyA)/BlyB tvori transmembranske pore in tako opredeliti njuno biološko vlogo. Za biokemijsko opredelitev proteina BlyB smo pripravili rekombinantni plazmidni vektor z zapisom za delecijsko mutanto BlyB (Δ25BlyB). Plazmidna vektorja z zapisoma za BlyA in BlyB smo transformirali v kompetentne celice E. coli BL21(DE3). Transformante smo dobili v obeh primerih. Protein BlyA smo uspešno izolirali in očistili z Ni-afinitetno kromatografijo ter izvedli preizkus lipidne specifičnosti s površinsko plazmonsko resonanco. Potrdili smo že predhodno dokazano, da se egerolizin BlyA močno veže na liposome, ki vsebujejo lipidne komplekse sfingomielin/holesterol (Hol), ne pa tudi na liposome, ki vsebujejo lipidne komplekse ceramid fosfoetanolamin/holesterol/fosfatidilholin (CPE:PC:Hol). Rekombinantnega proteina Δ25BlyB kljub večkratnim poskusom izolacije nismo uspeli pridobiti. Možni razlogi za neuspeh so lahko toksičnost proteina za celice E. coli ali nestabilnost proteina oziroma njegove mRNA. Within this thesis, we aimed to obtain the soluble recombinant protein beauveriolysin B (BlyB) in the bacterium Escherichia coli. In addition, we sought to determine whether the protein pair beauveriolysin A (BlyA)/BlyB forms transmembrane pores, thereby defining their biological role. For the biochemical characterization of BlyB, a recombinant plasmid vector encoding a deletion mutant of BlyB (Δ25BlyB) was constructed. Plasmid vectors encoding BlyA and BlyB were transformed into competent E. coli BL21(DE3) cells, and transformants were obtained in both cases. The BlyA protein was successfully isolated and purified using Ni-affinity chromatography, and its lipid specificity was analyzed by surface plasmon resonance. We confirmed previous findings that the aegerolysin BlyA binds strongly to liposomes containing sphingomyelin/cholesterol (SM:Chol) lipid complexes, but not to liposomes containing ceramide phosphoethanolamine/cholesterol/phosphatidylcholine (CPE:PC:Chol). Despite multiple attempts, the recombinant protein Δ25BlyB could not be obtained. Possible reasons for this failure include the toxicity of the protein to E. coli cells or the instability of the protein or its mRNA. Egerolizin protein z domeno MACPF beauveriolzin Beauveria bassiana umetne lipidne membrane rekombinantni protein Aegerolysin MACPF domain protein beauveriolysin Beauveria bassiana artificial lipid membranes recombinant protein true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-03-19 07:16:27 2026-03-19 07:16:32 2026-03-22 03:50:04 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 279146 272314627 56947.pdf 56947.pdf 1 A1F3EE613A4911232FD561513021D782 f3430307b632cafdc36b6e5aa317e275ff4adbe80595bf532ae60ae2bba375a6 1a987ac0-235b-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=230834 Biotehniška fakulteta 0 0 0