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Recombinant preparation of isotopically labeled receptor rNKR-P1A for NMR studies
Čonka, Martin ; Bezouška, Karel (advisor) ; Novák, Petr (referee)
anglicky NK cells belong to the population of cells which are able to cytotoxic kill certain tumor and cells infected by viruses. This bachelor thesis focuses on the rat's NK cell receptor, especially rNKR-P1A. This protein belongs to the activating receptors and is able to activate the cytotoxic function of NK cells. Physiological ligand and three-dimensional structure of this protein has not been resolved yet. The aim of this work was optimalization of production rNKR-P1A in minimal medium and thereafter producing isotope-labeled proteins. Isotope- labeled proteins could contribute to solving the three-dimensional structure of this rat receptor by using NMR methods.
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Characterization of transgenic forms of dipeptidylpeptidase IV expressed in astrocytoma cell line U373MG
Vomelová, Ivana ; Bezouška, Karel (advisor) ; Vaníčková, Zdislava (referee)
Dipeptidyl peptidase IV (DPP-IV) is a serine protease, which executes its proteolytic activity by cleaving X-Pro dipeptides from the N-termini of its substrates. Furthermore, DPP-IV exhibits many biological functions independent of its enzymatic aktivity. Previous studies in our laboratory proved increased expression of DPP-IV in high-grade astrocytic tumours. To evaluate the enzymatic and non-enzymatic functions of DPP-IV in a glioma model, clones of asctrocytic cell line U373MG transfected by enzymatically inactive, mutated DPP-IV (mutDPP-IV) and enzymatically active, wild type DPP-IV (wtDPP-IV), were prepared. Enzymatically inactive mutDPP-IV was prepared using point mutation the active site serine residue. Cells U373MG were transfected using a doxycycline inducible Tet-On® system. For further analysis of the transgenic forms of DPP-IV, methods were used for verification of protein expression, enzymatic activity and subcellular localization. Doxycycline induced U373MG mutDPP-IV and U373MG wtDPP-IV cells, expressing mutated and wild type DPP-IV, respectivelly, exhibited increased expression of transgenic DPP-IV in a concentration and time dependent manner. Doxycycline induced U373MG wtDPP-IV cells exhibited both increased expression and enzymatic activity of DPP-IV. In contrast, DPP-IV enzymatic...
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Recombinant expression and studies on DCL-1, receptor of dendritic cells
Pospíšilová, Eliška ; Bezouška, Karel (advisor) ; Novák, Petr (referee)
Recombinant expression and studies on DCL-1, receptor of dendritic cells Eliška Pospíšilová Charles University in Prague, Faculty of Science, Department of Biochemistry ABSTRACT Dendritic cells, which could be found in large numbers in many tissues, exprime C-type lectine receptor DCL-1 (in CD nomenclature CD302) on their surface. This molecule belongs to the class one transmembrane proteins. N-terminal sequence a C-type lectin-like domain (CTLD) it has on the extracellular side. Whereas the C-terminus, which contains potential phosphorylation site and signals for intracellular transport, is in the cytosol. As the sequence of this protein is highly conserved through all mammalian species, it is likely to play an important role in the immune system. Despite this fact, the DCL-1 molecule is still poorly investigated. This work deals with the extracellular part - especcially with the CTLD containing part - of the DCL-1 receptor. To learn more about this molecule, we attempted its recombinant expression in bacteria. We used pET-30a(+) based vector pDCL1E, and bacterial cells E. coli BL21 (DE3) Gold, which produced the target protein in the form of inclusion bodies into the cytoplasm. Therefore conditions for in vitro renaturation were optimalized, and it was prooved that the protein had native conformation...
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New possibilities of nitrilases in biocatalysis and bioremediation
Veselá, Alicja Barbara ; Bezouška, Karel (advisor) ; Weignerová, Lenka (referee)
Nitrilases are enzymes which catalyze the hydrolysis of nitriles to corresponding carboxylic acids. These enzymes have a great potential in biocatalysis, for example in the synthesis of mandelic acid and mandelamide, because of their chemo- and enantioselectivity. As bioremediation agents they are also applicable to sites contaminated with organic nitriles. In this work, activities of recombinant strains of E. coli expressing hypothetical nitrilases from fungi Giberella moniliformis and Nectria haematococca mpVI 77-13-4 were studied, as well as the biodegradation potential of bacteria from Rhodococcus and Nocardia genera towards benzonitrile herbicides dichlobenil (2,6-dichlorobenzonitrile), ioxynil (3,5-diiodo-4- hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydroxybenzonitrile). The hypothetical fungal nitrilases were expressed as functional enzymes. Nitrilase from G. moniliformis showed highest activity towards benzonitrile (30.9 U/mg protein), total activity yield was 2,560 U/l cell culture. The preferred substrate of the nitrilase from N. haematococca was phenylacetonitrile (12.3 U/mg prot.), total activity yield was 28,050 U/l cell culture. Nitrilase from N. haematococca was also able to hydrolyze mandelonitrile (5.9 U/mg prot.). Soil bacteria Rhodococcus rhodochrous PA-34, Nocardia globerula...
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Immunomodulation using two types of ligands
Adámek, David ; Bezouška, Karel (advisor) ; Pompach, Petr (referee)
CD69 and NKR-P1 are receptors belonging to C-lectin family of receptors. We have recently identified two types of effective ligands for these receptors: dimeric N-acetyl-D-glukosamines (activators) and (calix[4]arenes) inhibitors. By using them, we can modulate immune response. First class of ligands (N-acetyl-D-glukosamines) is able to bind to CD69 and NKR-P1 and activate the cells. Second class of ligands (calix[4]arenes) is able to effectively inhibit functions of CD69, but not functions of NKR-P1. The aim of this study was to learn how to modulate immune response by various combinations of these compounds, and to explore their concentrations suitable for tumor therapies. Effect of used ligand and their combinations was studied in standard tests of cell proliferation, cell death and cytotoxic killing.
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