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.
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