| Abstract [eng] |
The aims of the study were to determine the reasons for unsuccessful expression of measles virus hemaglutinin (MeH) in the yeast cells and to generate a stable yeast strains with integrated genes of protein secretory pathway of human cells and to examine influence of coded human proteins on MeH maturation. For the firs time, overexpression of MeH in yeast S. cerevisiae and P. pastoris was described. It was demonstrated that mechanisms of cotranslational translocation into the endoplasmic reticulum (ER) and protein maturation in the ER of yeast cells are not adapted to deal with for such complex virus glycoproteins. Proteomic analysis revealed, that overexpression of human virus surface protein precursors induces cytosolic unfolded protein response (UPR-cyto) in the yeast S. cerevisiae. A key feature of this response is the formation of extremely large aggregates involving macromolecular structures of eEF1A. Efficient mammalian like cotranslational translocation pathway was attempted to reconstitute in yeast cells by transferring human SRP, Sec61 complexes and TRAM1 protein. Human chaperones BiP, clanexin, calreticulin, ERp57 and PDI were transferred to the yeast cells to create suitable environment for maturation of MeH in the ER. Even though yeast strains, able to produce biologically active MeH protein, were not generated during this study, results show, that humanization of yeast secretory pathway, designed for producing active virus glycoproteins, is possible. |
