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Our group is involved in studies of function of proteins in CD44 and PTCH-GLI pathways playing the role in the formation and progression of cancer. Our studies show that recombinant CD44 having antiangiogenetic properties can be used in cancer treatment. We found also that GLI1 has potential as a diagnostic marker and as a target for antitumor drugs, thus antibodies against GLI1 have potential in cancer diagnostics and therapy. Biological as well as at the later stage clinical experiments require rather large amounts of those proteins in their native form. For production of proteins in their native form we have to use mammalian cells producers. As we have found preliminary that mammalian cells can be grown succesfully in serum-deprived media with yeast extract as an growth promotive component, the main direction of this project is the study of the effect of yeast extract on cultured mammalian cells. Aims of current project are: 1) the study of yeast extract on growth peculiarities of mammalian cell producers for optimisation of laboratory-scale production processes of recombinant proteins and antibodies; 2) the development of inexpensive serum-and protein-free media for laboratory-scale cultivations; 3) the comparative study of the effect of different yeast extract preparations; 4) the development and optimisation of purification processes of recombinant proteins and antibodies produced in bacteria, insect or mammalian cells.This work will involve the study of growth (growth rate, viability, cell cycle distribution, apoptosis) and production parameters of mammalian cells growing on serum-free media supplemented with yeast extract. The effect of yeast extract on growth promotion, sustained growth and apoptosis of cultured mammalian cells will be studied. We will attempt to identify the growth-promoting and growth-inhibiting compounds from yeast extract using MALDI-MS analysis of yeast extract fractions. The finding of growth promoting factor could give the possibility to control growth rate, cell density and production of recombinant proteins in insect and mammalian cell cultures and to design chemically defined media for those cells. Recombinant proteins for biological and clinical experiments will be purified using a combination of techniques such as ion exchange chromatography, hydrophobic chromatography and gelfiltration. One-step purification methods using specific tags will be also used. |