20.500.12556/RUL-101845 Pridobivanje ter karakterizacija rekombinantnega egerolizina in proteina z domeno MACPF iz entomopatogene glive Beauveria bassiana. Production and characterisation of aegerolysin and protein with MACPF domain from entomopathogenic fungus Beauveria bassiana. Perforini so proteini, ki s tvorjenjem por v membrane predstavljajo velik biotehnološki potencial na biofarmacevtskih, analitičnih in agronomskih področjih. Znanih je več različnih perforinov in proteinskih parov, ki skupaj tvorijo pore, njihov širok spekter možne uporabe pa kliče po novih raziskavah. Pomembno je natančno poznavanje njihovih molekularnih mehanizmov, ki vplivajo na njihovo biološko funkcijo in možne aplikacije. V tej diplomski nalogi smo se odločili preveriti potencial entomopatogene glive Beauveria bassiana, njenega egerolizina in proteina z domeno MACPF, z namenom ugotavljanja njune interakcije pri formiranju por. Izbrali smo dva gena, za katera je obstajala verjetnost, da glivi omogočata entomopatogeno delovanje s tvorbo por v žuželčjo kutikulo. Tekom raziskave smo uspešno klonirali in izrazili oba proteina v rekombinantni bakteriji, egerolizin smo uspešno izolirali v čisti obliki. Na njem smo uspešno izvedli več analitskih metod, ki so pokazale, da smo protein najverjetneje pridobili v pravilnem zvitju. Sedimentacijski test in površinska plazmonska resonanca sta pokazala močno vezavo egerolizina na kombinacijo lipidov ceramid fosfoetanolamin in sfingomielin, kar nakazuje na predvideno biološko funkcijo. Za natančnejšo določitev bioloških funkcij obeh proteinov in dokončno preverbo njunih potencialov kot pesticidnih sredstev in morebitne druge aplikacije pa so zagotovo potrebne nadaljnje analize. Perforins are proteins that have a large biotechnological potential in biopharmaceutical, analytic and agronomical fields because of their pore forming abilities. There is already a wide spectre of known perforins and protein couples that jointly form pores, but their wide spectre of potential use calls for new research. Analysis of molecular mechanisms that effect their biological function as well as their potential applications is crucial. In this thesis we decided to discover potential of entomopathogenic fungi Beauveria bassiana, its aegerolylin and protein with MACPF domain with a goal of discovering their interaction in formulation of pores. We selected two genes, for which there was a plausibility, that they are enabling fungi its entomopathogenic activity, with formation of pores into insect cuticle. During the research we successfully cloned and expressed both proteins from recombinant bacterium and successfully isolated aegerolysin in a pure form. On this protein we than preceded to conduct analytical methods, that confirmed, that we most likely gained it in its native form. Sedimentation test and SPR showed strong bonds of aegerolysin to lipid combination of ceramide phosphoethanolamine and sphingomyelin which matches predicted biological function. For exact determination of biological functions of both proteins and final discovery of their potential use as pesticide and other applications further research is needed. biotehnologija entomopatogene glive egerolizini domena MACPF perforini biotechnology aegerolysin entomopathogenic fungi MACPF domain perforins true false false [Š. Lemež] Slovenski jezik Angleški jezik Diplomsko delo/naloga 2018-07-08 07:46:19 2018-07-08 07:46:21 2022-08-15 03:38:50 0000-00-00 00:00:00 2018 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 632.937.14:601.4:577.12(043.2) 151515 9006969 3359.pdf 3359.pdf 1 A51A3BCC98589579B3D0413A391767CF 536473316fb5830555f625767673217a3ec7128431bdeda4a8e1738f0d69029d 6b5a7c18-a1b5-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=112060 Biotehniška fakulteta 0 0 0