EpCAM (Epithelial Cell Adhesion Molecule) is a transmembrane glycoprotein recognized as a tumor antigen. It plays a key role in cell proliferation, differentiation and is involved in many signaling pathways. RIP (regulated intramembrane proteolysis) causes degradation of EpCAM. Initial cleavage results in the soluble extracellular domain (EpEX) which is a ligand for EGFR (Epidermal Growth Factor Receptor). Previous attempt to characterize the EpEX-EGFR complex was unsuccessful. We hypothesize that post-translational modifications have a crucial role in the interaction between EpEX and EGFR, so we designed 4 different constructs for the expression of EpEX in mammalian cell lines (CHO and HEK), because the glycosylation and other post-translational modifications in insects can differ from the one in mammals. In two of them, we integrated albumin signal peptide instead of native one to improve secretion efficiency. Two constructs include WPRE (Woodchuck hepatitis virus Post-transcriptional Regulatory Element) which enhances transgene export of mRNA from the nucleus to the cytoplasm. Initially, we attempted to generate stable cell lines for the expression of recombinant EpEX and to compare the level of recombinant protein expression between all four constructed vectors. But we ended up with successfully expressed recombinant EpEX in transiently transfected cells Flp-In™ T-REx™-293. In future experiments, we would aim to produce stable cell lines using the Flp-In™ system and quantitatively analyze recombinant protein expression. Our work provides the basis for the expression of larger amounts of EpEX in mammalian cell lines. This will aid to elucidate the effect of post-translational modifications on EpEX-EGFR interaction.
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