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

Protein EGFR je transmembranski glikoprotein, ki spada v skupino tirozin kinaznih receptorjev in ima pomembno vlogo pri proliferaciji, diferenciaciji ter signalizaciji celice. Velik vpliv pri pravilni signalizaciji proteina EGFR imajo post-translacijske modifikacije, predvsem pravilna glikozilacija. Da bi okarakterizirali vpliv glikozilacije EGFR na vezavo liganda EpEX, smo protein EGFR izrazili v sesalskih celičnih linijah z uporabo sistema Flp-In, ki omogoči, da se željeni gen vključi na točno določeno mesto v genomu. Pripravili smo 4 različne zapise za zunajcelični del EGFR (EGFR-Ex) in sicer z nativnim signalnim peptidom z in brez elementa WPRE ter z albuminskim signalnim peptidom z in brez elementa WPRE. S tem smo želeli preveriti vpliv albuminskega signalnega peptida ter elementa WPRE na nivo izražanja EGFR-Ex, saj naj bi oba prispevala k povišanemu izražanju rekombinantih priteinov. Transfekcijo smo izvedli v celicah Flp-In™-CHO in Flp-In™-293. Izražanje proteina EGFR smo s prenosom western ter imunodetekcijo potrdili pri vseh štirih konstruktih v celicah HEK, kjer je izražanje potekalo prehodno. Pri celicah CHO, kjer je potekalo stabilno izražanje vseh štirih konstruktov, pa smo uspeli potrditi le izražanje konstrukta z nativnim signalnim peptid ter elementom WPRE v samem vektorju. Kvantitativne analize vpliva albuminskega signalnega peptida in elementa WPRE na nivo izražanja EGFR-EX nam žal ni uspelo izvesti.The EGFR protein is a transmembrane glycoprotein that belongs to the group of tyrosine kinase receptors and plays an important role in cell proliferation, differentiation and signalling. Post-translation modifications, especially the correct glycosylation, have tremendous influence with regards to the signalization of the EGFR protein. To characterize the effect of EGFR glycosylation on EpEX ligand binding, EGFR protein was expressed in mammalian cell lines CHO and HEK293 using the Flp-In system, which allows the desired gene to integrate at a specific spot in the genome. We prepared 4 different constructs for the extracellular part of EGFR (EGFR-Ex)one with a native signal peptide with the WPRE element, the second one with a native signal peptide, but without WPRE element, the third one with an albumin signal peptide with the WPRE element, and the fourth one with an albumin signal peptide without the WPRE element. The goal was to check the influence of albumin signal peptide and the element WPRE on the level of EGFR-Ex expression, as both are thought to contribute to the increased expression of recombinant proteins. The expression of the EGFR protein was confirmed by western blot and immunodetection in all four constructs in HEK cells where the expression was with transient transfection. In stable cell line CHO, we were able to confirm only the expression of the construct with the native signal peptide and the WPRE element in the vector itself. Quantitative analysis of the influence of albumin signal peptide and WPRE element on the level of EGFR-EX expression was unfortunately not performed