Priprava proteinov Wnt in test interakcije Wnt:EpCAM

Struna, Gašper (avtor), Pavšič, Miha (mentor)

Wnt proteini so glikoproteini, ki imajo vezan palmitoleat in so pomembni pri proliferaciji celic in raku. Pri človeku sodelujejo pri več signalnih poteh, ki se delijo na kanonične in nekanonične signalne poti. Pri kanonični signalni poti je pomemben transkripcijski regulator β-katenin, na katerega se med drugim lahko veže tudi znotrajcelični del tumorskega označevalca EpCAM. Zunajcelični del EpCAM (EpEX) vsebuje hidrofobni žep, v katerega se lahko veže palmitat. Ker se proteini EpCAM in proteini Wnt signalne poti v literaturi omenjajo pogosto v istem kontekstu, smo želeli nekaj izbranih proteinov izraziti v celicah HEK293T in preveriti, ali jih lahko veže EpEX preko svojega hidrofobnega žepa. Celice HEK293T smo pri različnih pogojih transfecirali s plazmidi, ki so vsebovali zapise za Wnt proteine z oznako V5. Po transfekciji smo izvedli test »pull down« z Wnt proteini in nemutirano oziroma mutirano (zaprt žep) obliko EpEX. Obe obliki smo predhodno pripravili v insektnih celicah in očistili z nikljevo afinitetno kromatografijo. Rezultati po transfekcijah so pokazali, da se Wnt proteini v celicah izražajo v majhnih količinah. Na membrani po testu »pull down« in sledečem western prenosu nismo zaznali nobene lise. Vzrok neprisotnosti lis bi bila lahko majhna količina izraženih Wnt proteinov zaradi česar signala ne moremo zaznati. Druga možnost pa je, da interakcije med Wnt in EpEX ni ali pa je ta šibka. V nadaljnjih študijah bi bilo torej potrebno optimizirati pogoje transfekcije in ponovno preveriti potencialno interakcijo med EpEX in Wnt.

• Jezik: Slovenski jezik
• Ključne besede: Wnt, transfekcija, EpEX, interakcija
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2023
• PID: 20.500.12556/RUL-149437
• COBISS.SI-ID: 169094403
• Datum objave v RUL: 07.09.2023

STRUNA, Gašper, 2023, Priprava proteinov Wnt in test interakcije Wnt:EpCAM [na spletu]. Diplomsko delo. [Dostopano 18 april 2025]. Pridobljeno s: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=slv&id=149437

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Preparation of Wnt proteins and test of Wnt:EpCAM interaction

Izvleček

Wnt proteins are glycoproteins that have palmitoleate attached and are important in cell proliferation and cancer. In humans, they participate in several signaling pathways, which are divided into canonical and non-canonical signaling pathways. In the canonical signaling pathway, the transcriptional regulator β-catenin is important, to which, the intracellular part of the tumor marker EpCAM can bind. The extracellular part of EpCAM (EpEX) contains a hydrophobic pocket into which palmitate can bind. As EpCAM proteins and Wnt signaling pathway proteins are often mentioned in the literature in the same context, we wanted to express some selected proteins in HEK293T cells and check whether EpEX can bind them via its hydrophobic pocket. HEK293T cells were transfected under different conditions with plasmids containing transcripts for V5-tagged Wnt proteins. After transfection, we performed a pull-down assay with Wnt proteins and the unmutated or mutated (closed pocket) form of EpEX. Both forms were previously prepared in insect cells and purified by nickel affinity chromatography. The results after the transfections showed that Wnt proteins are expressed in small amounts in the cells. We did not detect any band on the membrane after the pull-down assay and the following Western blot. The reason for the absence of band could be a small amount of expressed Wnt proteins, due to which the signal cannot be detected. Another possibility is that the interaction between Wnt and EpEX is absent or weak. In further studies, it would therefore be necessary to optimize the transfection conditions and recheck the potential interaction between EpEX and Wnt.

• Ključne besede: Wnt, transfection EpEX, interaction