| Abstract [eng] |
Growing market of the glycoprotein based drugs increases demands of safe, cheap and effective expression systems for production of glycoproteins. Mammalian cell cultures, which are being used for this purpose, are very expensive and ineffective. Yeasts are rising as one of the best alternatives. Well known genetics, biochemistry and physiology are only few advantages. Yeasts are also considered to be safe and easy to manipulate organism. Still, despite introducing humanized glycosylation pathways, yeast based expression systems are not able to produce glycoproteins for pharmaceutics, with a very few exceptions. So, further researches in adapting yeast for glycoproteins synthesis must be made. This work is directed for this purpose. In this work mumps, measles and influenza virus surface glycoproteins were expressed in yeast Pichia pastoris. Results show, that mumps virus hemagliutinin-neuraminidase is not synthesized in P.pastoris. Synthesis of measles virus (MeV) hemagliutinin (H) glycoprotein was not effective, with recombinant protein not possessing characteristics of native analogue. MeV-H was found in two forms: unglycosylated polypeptide precursor and glycosylated form, both aggregated and insoluble in non-ionic detergent. Increase in MeV-H expression level resulted in extensive accumulation of unglycosylated MeV-H protein precursors in the cytoplasm of yeast cells. Addition of S.cerevisiae α-factor secretion signal sequence to globular part of MeV-H made translocation through endoplasmic reticulum (ER) more effective, but recombinant protein was not secreted in culture media. Contrarily, influenza virus (InV) neuraminidase (NA) was secreted in culture media, confirming previously shown results. Recombinant NA protein showed antigenic properties similar to its native analogue. However, the major part of the recombinant NA was found inside the cell in glycosylated non soluble aggregates. Accumulation of both MeV-H and InV-NA glycosylated forms led to the cell lysis. Synthesis of MeV-H induced significant changes in cells proteome. Unglycosylated and glycosylated forms stimulated expression of different proteins. Glycosylated form exceptionally increased synthesis of BiP (ER chaperone), MANA (Mannose-6-phosphate isomerase) and BGL2 (Glucan-1,3-β-glucosidase), which were identified using 2-dimensional electrophoresis and bioinformatical methods. Increase in MANA and BGL2 production, which shows disorders in glycoproteins synthesis and cell biogenesis respectively, explains the cell lysis, mentioned above. Yeast cell lysis resulted in appearance of bead-shaped derivatives, formed from cell wall, which were about 1µm in diameter and could be observed under microscope. These beads were easily purified and could be used for determining of yeast cell wall composition and properties. Results of this work reveal the processes leading to formation of insoluble and inactive aggregates of recombinant measles virus hemagglutinin in yeast cells and show why complex human glycoproteins are not efficiently produced in yeast. The reason of inefficient virus surface protein expression in yeast lies on different protein maturation processes in mammalian and yeast cells, comprising the early stages of cell secretion pathway: translocation across the ER membrane and protein folding in the ER lumen. This could be the guideline improving yeast expression systems for effective synthesis of active human virus surface glycoproteins. |
