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Optimization of HEK293 cell line expression system by regulation of cell cycle and apoptosis
Poláchová, Edita ; Vaněk, Ondřej (advisor) ; Pavlíček, Jiří (referee)
Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...
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Production and purification of recombinant receptor Clrb
Prokopová, Tereza ; Vaněk, Ondřej (advisor) ; Černá, Věra (referee)
The Natural Killer (NK) cells play a vital role in the nonspecific immunity. They are capable of efficient immunity reaction without any antigen specific receptors on their surface. NK cells recognize non-self molecules and they also recognize their molecules serving as health markers, and absence of these molecules such as MHC glycoproteins on the target cell surface. The NK cells are able to recognize viral infection or tumor transformation in the organism. If natural killer cell is in contact with a cell carrying an abnormally low MHC class I glycoproteins, it will create a signal which informs the cell is infected with a virus. NK cells trigger apoptosis of the target cell without prior stimulation, proliferation and differentiation. They also promote inflammatory responses by the production of chemokines and cytokines. The response is always the interplay of activating and inhibitory signals that the cell receives from its surroundings. The latest research shows that the targeted modulation of NK cells leads to less complications in bone marrow transplantation. They can be potentially used in immunotherapy, e.g. in the treatment of autoimmune diseases. Therefore, NK cells are a highly-studied group of cells. This thesis is focused on a production of Clrb ("C- type-lectin-related protein b")....
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