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Comparison of biotechnological procedures for pure proteins preparation
Bušanski, Patrik ; Langová, Denisa (referee) ; Brázda, Václav (advisor)
The production of recombinant proteins is a biotechnological process during which proteins are produced in foreign organisms by gen manipulation. To form a recombinant plasmid the gene encoding the desired protein is isolated and inserted into an expression vector. The plasmid is then transformed using physical or chemical method into a suitable host, where the recombinant gene is translated into amino acid sequence in the newly synthetized protein. The theoretical part of this bachelor thesis includes characteristics of proteins, methods of recombinant protein preparation and compares individual expression systems. Three isoforms of the p53 protein, which were synthesized in the E. coli microorganism, were selected for processing the experimental part. The transformed recombinant plasmid contained two tags for purification, HIS-tag and GST-tag, making it possible to compare the efficacy of the two purification methods. HIS-tag purification was found to work for all three isoforms better, with concentrations of recombinant proteins were several times higher than those of the GST-tag. The p53 proteins are about 50 kDa long, what was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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Dominant protein antigens of Toxocara canis
Skulinová, Kateřina ; Kašný, Martin (advisor) ; Panská, Lucie (referee) ; Vadlejch, Jaroslav (referee)
Larval toxocarosis is a worldwide widespread zoonosis occurring in developed countries as well as developing countries. The disease is caused by roundworms of the genus Toxocara, primarily intestinal parasites of dogs, cats and other animals. Viable eggs released into the environment with the dog's faeces can infect not only definitive hosts, but also paratenic hosts, which include many vertebrates, some invertebrates, and also humans. In humans, larval migration can cause severe and irreversible tissue damage, which is characterized by various clinical forms of the disease. For the purposes of routine diagnosis of larval toxocarosis, the most frequently used method so far is ELISA and Western blot, which enable the reaction demonstration of specific antibodies with the larval excretory-secretory product (TES). TES is obtained for diagnostic purposes from larvae cultured in nutrient medium. The preparation of such an antigenic mixture is very laborious and may vary across the laboratories. Current research in the field of diagnosis of larval toxocarosis is therefore focused on the standardization of serodiagnostic procedures. A fundamental prerequisite is knowledge of the detailed composition of TES, especially antigenic (protein) molecules. However, the number of studies devoted to the...
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Optimization of culture conditions of selected {E.coli} strains producing recombinant proteins
MICHALCOVÁ, Zuzana
Escherichia coli is one of the most preferred organisms to produce recombinant proteins. E. coli is a well-established host that offers ease of genetic manipulation, short culture times, continuous fermentation capability and affordable culture media. To achieve high levels of protein expression, and therefore high recombinant protein production, optimization of culture conditions is required. This thesis is focused on the optimization of cultivation, the practical part is focused on the production of Uridine Phosphorylase (UP) and Purine Nucleoside Phosphorylase (PNP) enzymes. E. coli bacteria producing UP or PNP were cultured on six media with different nutrient sources. Protein production was verified on a spectrophotometer by the Bradford assay and further by polyacrylamide gel electrophoresis with SDS (SDS-PAGE). Enzyme activity was measured by HPLC assay. In addition, the dry weight biomass was determined for all samples. Based on results from these assays, Terrific Broth medium with glucose addition after 4 h of cultivation (TBG4) was selected and subsequently used for upscaling to the fermenter. Successful cultivation in the fermenter confirmed the results of cultivation in Erlenmeyer flasks.
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Příprava mutantního serpinu z klíštěte \kur{Ixodes ricinus}
EDEROVÁ, Monika
Point mutation altering arginin for tryptophan amino acid residue in P1 site of tick salivary serpin Iripin-1 was created using specific primers. Recombinant protein with this mutation in nucleotide sequence was then expressed in chemically competent Escherichia coli cells, extracted from them and purified by affinity and size-exclusion chromatography. To see the impact of the mutation on inhibitory function of Iripin-1, its ability to bind trypsin and form covalent complexes was evaluated.
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Roles of tubulin post-translational modifications in regulation of microtubule-based processes
Šliková, Pavlína ; Novák, Petr (advisor) ; Libusová, Lenka (referee)
Microtubular cytoskeleton plays crucial roles during diverse cellular processes, such as intracellular transport, cell motility and chromosome segregation during cytokinesis. Tubulin, the building block of microtubules, undergoes numerous post-translational modifications which affect microtubular dynamics and organization as well as their interaction with associated proteins. Understanding the role post-translational modifications play in the diversification of functions and properties of microtubules is key for our comprehension of the dynamics of the complex microtubule cytoskeleton. However, mechanisms behind the effect of post-translational modifications on microtubule cytoskeleton are not fully understood. In this work, we focus on the influence of post-translational modifications on microtubule polymerization and interaction with molecular motor kinesin-1. Using total internal fluorescence and interference reflection microscopy techniques, we here show that high levels of post-translational modifications on microtubules decrease the time of microtubule-kinesin interaction whereas binding affinity and median velocity are not significantly different on modified and unmodified microtubules. Further, we show that the absence of polyglutamylation on tubulin isotypes leads to a faster microtubule...
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Preparation and characterization of human cellular cofactors of retroviral integration.
Čermáková, Kateřina ; Maloy Řezáčová, Pavlína (advisor) ; Obšil, Tomáš (referee)
Lens epithelium-derived growth factor/p75 (LEDGF/p75) is a prominent cellular binding partner of Human Immunodeficiency Virus type 1 (HIV-1) integrase. It is a human nuclear protein, which has been implicated in transcriptional regulation and cell survival. The role of LEDGF/p75 in HIV integration is well characterized, the HIV integrase binding domain (IBD) was identified and structural studies, which provide detail information about this interaction, were done. However, very little is known about its physiological function. As a transcriptional co-activator, LEDGF/p75 is implicated not only in HIV replication, but also in human cancer and autoimmunity. Key feature for both, the viral and cellular role of this protein, is its ability to act as a molecular adaptor tethering proteins to the chromatin fiber. Recently, PogZ (Pogo transposable element derived protein with zinc finger domain) was identified and validated as a new cellular interaction partner of LEDGF/p75. It was shown, that their interaction is mediated by IBD of LEDGF/p75 and the C-terminal domain of PogZ. To gain more insight in this interaction, we have initiated structural studies of their complex. Structural information is crucial for understanding the LEDGF/p75 biological role and might help in design of inhibitors selectively blocking...
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Study of receptor-ligand pair NKR-P1F and Clrg
Kotýnková, Kristýna ; Man, Petr (advisor) ; Schneider, Bohdan (referee)
Study of receptor-ligand pair NKR-P1F and Clrg Mouse NKR-P1F:Clr-g receptor:ligand pair is important component of the receptor "zipper" that occurs at the contact between natural killer cell and its target cell, and represents a recently discovered example of lectin-lectin interactions important for recognition among immune cell subsets. In order to study structure of these proteins and interactions between them, we have prepared pET-30a(+) bacterial expression vectors coding parts of extracellular domains of the two receptors. After induction of protein production with IPTG, the proteins precipitated into inclusion bodies, from which they could be refolded in vitro. Refolded proteins were purified using combination of ion exchange and size exclusion chromatography. NKR-P1F construct yielded only small amounts of soluble protein using standard refolding protocols. Furthermore we have experienced difficulties with reproducibility of the refolding results. In the case of Clrg the standard protocols for protein refolding were not sufficient. In order for the Clrg to fold properly, the odd cysteine which does not fit into the pattern usual for this family of receptors was substituted for serine and resulting C148S construct was shown to be more useful. Further, using (benzyldimethylammonio)propanesulfonate in...
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Influence of cultivation conditions on the production of recombinant proteins
Gardošová, Zuzana ; Nováčková, Ivana (referee) ; Brázda, Václav (advisor)
Recombinant proteins are produced by using genetic modifications. In this process is insert contained gene encoding a certain protein in a cloning vector cloned into the host organism. Recombinant proteins are expressed after the transformation of the cloning vector into a host, the host organism. The expression of recombinant proteins in bacterial cells is one of the most efficient ways to manufacture these proteins. The p53 protein is a tumor suppressor protein, the main role in cells is to react on DNA damage. Due to the reaction to various intracellular and extracellular stimuli, including DNA damage, the p53 protein shows different biological functions, including regulation of senescence, cell cycle, or apoptosis. The theoretical part of the thesis part presents the basic properties of proteins, methods of recombinant protein expression, methods of protein isolation, and characterization of p53 protein. The aim of the experimental part was to determine the effect of incubation temperature on recombinant p53 protein production. The work involves the isolation of plasmid DNA and its transformation into E. coli production cells. The produced proteins were successfully isolated and subsequently characterized by SDS-PAGE and Western Blotting.
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Structure and dynamics of mouse C-type lectin-like receptors.
Wallenfels, Lucie
Natural killer (NK) cells represent indispensable part of the innate immunity as they are capable of promptly identifying virally infected or tumor cells and participating in the regulation of adaptive immune responses. These functions are ensured by the interplay between NK receptors, creating a complex regulatory system. Solving the receptors' structure may contribute to an overall understanding of NK cell biology. Presented thesis describes an elucidation of the structure of the inhibitory C-type lectin-like receptor (CTLR) Nkrp1b with an emphasis toward structural features (stalk, loop and oligomerization state) which might affect conformation or interactions of this receptor. The interaction of Nkrp1b with its ligand, Clr-b protein, is immunologically significant as it regulates NK cells' activity independently and monitors changes that are not visible to cytotoxic T lymphocytes. To study individual structural aspects of Nkrp1b, two protein variants were recombinantly prepared in bacterial expression system: entire ectodomain and ligand-binding domain lacking the stalk. Using a range of mass spectrometric techniques in combination with homology modeling and molecular dynamics, we proposed the Nkrp1b structure including its monomeric and dimeric arrangements. In addition, the oligomerization...
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