İnsan EpCAM'ın ekstrasellüler bölgesinin moleküler klonlanması ve rekombinant üretimi

Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein of approximately 40 kDa size that facilitates Ca2+-independent homotypic cell and cell adhesion in epithelia. EpCAM consist of two domains; extracellular domain (EpEX) and intracellular domain (EpICD), separated by a transmembrane helix. EpCAM's roles include cellular signaling and migration. The overexpression of EpCAM in epithelial tumors and downregulation in normal epithelia is used as a diagnostic tumor marker. Previous studies demonstrated that EpCAM can serve as an attractive anti-tumor target such as immunotherapy, RNA aptamers, and vaccination. Therefore, we aimed to express and purify recombinant EpEX molecules in an (E. coli) based expression system in order to use it in downstream applications. RNA isolated from two different human cancer lines highly expressing EpCAM was used to amplify the cDNA coding for EpEX. PCR amplified EpEX cDNA fragments were ligated into pGEX-6P1, a plasmid vector for bacterial expression and purification of recombinant proteins. The correct pGEX-6P1-EpEX clone was isolated and verified by colony PCR screening, restriction analysis, and subsequent Sanger sequencing. The plasmid was transferred to BL21 strain for expression of the recombinant protein. In order to obtain the highest yield and purity for recombinant EpEX molecule, induction, expression and purification conditions were optimized. GST-EpEX was purified by affinity chromatography utilizing glutathione-agarose beads. HRV3C protease was used to cleave and release the recombinant EpEX molecules from the GST tag that remains bound to the beads. And finally, the results were analyzed by Coomassie Blue staining of SDS-PAGE separated protein samples.