20.500.12556/RUL-184135 Priprava zunajceličnih delov potencialnih interakcijskih partnerjev EpCAM Preparation of extracellular domains of potential interaction partners of EpCAM EpCAM je transmembranski glikoprotein tipa I, ki sodeluje pri embrionalnem razvoju, delovanju epitelija ter celični proliferaciji, migraciji in adheziji. Poleg tega prispeva k razvoju in napredovanju raka, zato predstavlja pomemben tumorski označevalec in obetavno terapevtsko tarčo. EpCAM je vključen v različne signalne poti, med katerimi je najpomembnejša Wnt/β-kateninska signalna pot, ki je aktivirana prek regulirane intramembranske proteolize (RIP). Cepitev zunajceličnega dela EpCAM (EpEX), ki aktivira RIP, je najverjetneje regulirana s strani interakcij z drugimi proteini. Cilj diplomskega dela je bil pripraviti rekombinantne zunajcelične dele proteinov FZD7, EphB2 in MMP14, ki so bili identificirani kot potencialni interakcijski partnerji EpCAM in so morda z njim tudi funkcijsko povezani. V okviru diplomskega dela smo pripravili tri rekombinantne ekspresijske plazmidne vektorje, ki so vsebovali zapise za zunajcelične dele proteinov FZD7, EphB2 in MMP14, vključno z N-končnimi signalnimi peptidi in C-končnimi Strep-tag II oznakami. S pripravljenimi plazmidi smo prehodno transfecirali insektne celice High Five in uspešno izrazili rekombinantna zunajcelična dela proteinov FZD7 in MMP14. Poskusili smo ju tudi izolirati iz supernatantov celičnih kultur s Strep-Tactin afinitetno kromatografijo, vendar nam to ni uspelo, predvidoma zaradi nizkih ravni izražanja ali obarjanja pri dializi. EpCAM is a type I transmembrane glycoprotein involved in embryonic development, epithelial function, as well as cellular proliferation, migration, and adhesion. In addition, it contributes to cancer development and progression, making it an important tumor marker and a promising therapeutic target. EpCAM is involved in various signaling pathways, among which the most prominent is the Wnt/β-catenin signaling pathway, activated through regulated intramembrane proteolysis (RIP). The cleavage of the extracellular domain of EpCAM (EpEX), which activates RIP, is most likely regulated through interactions with other proteins. The aim of this diploma work was to prepare the recombinant extracellular domains of the proteins FZD7, EphB2, and MMP14, which have been identified as potential interaction partners of EpCAM and may also be functionally associated with it. As part of this diploma work, we constructed three recombinant expression plasmid vectors containing sequences for the extracellular domains of FZD7, EphB2, and MMP14, including N-terminal signal peptides and C-terminal Strep-tag II tags. Using the prepared plasmids, we transiently transfected High Five insect cells and successfully expressed the recombinant extracellular domains of FZD7 and MMP14. We also attempted to isolate them from cell culture supernatants using Strep-Tactin affinity chromatography; however, this was unsuccessful, presumably due to low expression levels or precipitation during dialysis. EpCAM potencialni interakcijski partnerji insektne celice izražanje rekombinantnih proteinov afinitetna kromatografija EpCAM potential interaction partners insect cells recombinant protein expression affinity chromatography true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2026-06-30 10:40:03 2026-06-30 10:40:15 2026-07-02 03:29:24 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 25679 Majerle_Nina_-_Priprava_zunajcelicnih_delov_potencialnih_interakcijskih_partnerjev_EpCAM.pdf Majerle_Nina_-_Priprava_zunajcelicnih_delov_potencialnih_interakcijskih_partnerjev_EpCAM.pdf 1 7183916D491011A8F40E6F8E9A35202A 7ebb7b3cf12cf36069b3ac7d809255b71cc38936f281a996ee40ce6897223b35 385d5c70-745f-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=236649 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0