Design of novel constructs for expression of recombinant EpCAM ectodomain in mammalian cell lines.

EpCAM (epitelijska celična adhezijska molekula) je transmembranski glikoprotein, ki so ga sprva prepoznali kot tumorski antigen. Ima pomembno vlogo v celični proliferaciji in diferenciaciji, vpleten je v številne signalne poti. RIP (nadzorovana intramembranska proteoliza) povzroči razpad proteina EpCAM. Začetna cepitev povzroči sprostitev topne zunajcelične domene (EpEX), ki je ligand receptorja epidermalnega rastnega faktorja (EGFR). Predhodno izvedeni poskusi karakterizacije kompleksa EpEX-EGFR so bili neuspešni. Naša hipoteza je, da imajo posttranslacijske modifikacije ključno vlogo v interakciji med proteinoma EpEX in EGFR, zato smo pripravili 4 različne vektorske konstrukte za izražanje proteina EpEX v sesalskih celičnih linijah (CHO in HEK), saj se lahko glikozilacija in druge posttranslacijske modifikacije v insektnih celicah bistveno razlikujejo od tistih v sesalskih celicah. V dva vektorja smo vključili albuminski signalni peptid namesto nativnega, da bi izboljšali učinkovitost izločanja proteina v zunajcelični prostor. Dva vektorska konstrukta vsebujeta WPRE (posttranskripcijski regulatorni element virusa WHV), ki poveča učinkovitost prenosa transgene mRNA iz jedra v citoplazmo. Prvotno smo skušali narediti stabilne celične linije za izražanje rekombinantnega proteina EpEX in primerjati stopnjo izražanje med vsemi štirimi pripravljenimi vektorskimi konstrukti. Vendar nam je uspelo uspešno izraziti rekombinanten EpEX le v prehodno transfeciranih celicah celične linije Flp-In™ T-REx™-293. V prihodnje bi z uporabo sistema Flp-In™ poskusili proizvesti stabilne celične linije in kvantitativno analizirati izražanje rekombinantnega proteina. Naše delo predstavlja osnovo za izražanje večjih količin rekombinantnega proteina EpEX v sesalskih celičnih linijah. To bi pomagalo pojasniti pomen posttranslacijskih modifikacij v interakciji EpEX-EGFR.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