EXPRESSION OF THE THREE PROTO-ONCOGENES, EGFR, MEK AND B-RAF AFTER TRANSFECTION INTO HUMAN CELLS AND THEIR DETECTION BY MASS SPECTROMETRY

Sammanfattning: When activated by growth factors and mitogens, cellular receptors like EGFR become activated and transmit signals from the cell surface through the MAPK pathway into the nucleus of the cell, in order to elicit a cellular response like growth, apoptosis, proliferation, and survival. Over activation of EGFR even without receptor binding and members of the MAPK pathways like BRAF and MEK1 have been reported in tumorigenesis. We used Gateway Technology to study the recombinant protein expressions from these 3 proto-oncogenes in HEK293T and HeLa cell lines, using pLenti 6.3/V5 as the expression system. BRAF was neither detected in the HeLa nor in the HEK 293T cell lines, by mass spectrometry after transfection. Only in the HeLa cell line was EGFR identified meanwhile MEK1 was identified both in HEK293T and HeLa cell lines. However, the results showed that the proteins identified were endogenous to these cell lines and therefore, no recombinant proteins were expressed using the pLenti 6.3/V5, as the expression system.