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Application of tissue culture test plates for production of recombinant protein in HEK293 cells; determination of optimal conditions
Krzyžanková, Marcela ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Efficient production of the recombinant proteins (r-proteins) must be based on previous testing of an expression of a small amount of the r-proteins. This work focuses on optimizing the expression of the r-proteins in 12-well plates. It includes testing of an appropriate speed of shaking, production and transfection volume. It compares all the current testing vessels (it compares a 50-ml centrifugation tube to new tested plates that can substitute the unsuitable tubes). It also compares these new tested plates to production square bottles in order to compare the r-protein expression in the plates to the r-protein expression in the bottles. It monitors effects of carbon dioxide on a number of vital cells, their viability, a relative frequency of positive cells on GFP in various cultivation vessels (plates, tubes, bottles), and pH of HEK 293 cellular cultivation during the 4-day cultivation process as well. On the basis of the results and statistical processing of the results, we have set the optimal agitation speed of 230 rpm for the 12-well plates. We have also set the appropriate production and transfection volume of 2 and 0.5 ml for the 12-well plates. In order to evaluate variables and compare cultivations in all the vessels, the tubes could be substituted by the plates. There is a statistically significant impact of carbon dioxide on the number of cells, their variability, relative frequency of cells (positive on GFP) and pH of the cellular HEK 293 cultivation in the cultivation vessels. There is the strongest r-protein expression in carbon dioxide conditions. The results of this work allow to employ the 12-well plates when we aim to test the expression of the r-proteins in a small amount and in carbon dioxide conditions. On the basis of the findings, the expression of the r-proteins in the 12-well plates and carbon dioxide conditions can substitute the expression of the r-proteins in the production bottle and in carbon dioxide free conditions.
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Transient transfection of a serum free cell culture using polyethyleneimines
Čutová, Michaela ; Španová, Alena (referee) ; Ševčík, Mojmír (advisor)
Master’s thesis deals with the transient transfection of the serum free animal cell culture using polyethyleneimines. In the theoretical part formation of recombinant DNA molecules, used expresion vectores, used DNA transfer and detection of recombinant proteins are discussed. The experimental part deals with efficiency of the polyethylenimine mediated transient transfection under various experimental conditions. 293HEK/EBNA cell line was chosen as an experimental model. First the most effective plasmide - pCEP4/SEAP was selected. Then three transfection methodes were tested: Muller (2005), Durocher et al. (2007) and Backliwal et al. (2008). The highest recombinant protein expresion was reached using the method of Backliwal et al. (2008).
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Cathepsin L of Sphaerospora molnari - localisation, function and diagnostic tools
NECHVILE, Rudolf Lukas
The aim of this thesis was to develop diagnostic and quantitative assays/tools based on western blotting, confocal microscopy and immunogold electron microscopy, as well as flow cytometry, to determine the localisation and potential function of the highly expressed cathepsin L of the myxozoan parasite Sphaerospora molnari (SmolCL) in its fish host, the common carp. Furthermore, a recombinant SmolCL was used in a vaccine trial to estimate its potential for raising antibodies in carp and test their immunoprotective potential towards S. molnari. This thesis provides new methodological tools for research and allows a greater understanding of myxozoan parasite-fish host interactions based on proteolytic enzymes.
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Preparation of antibodies to determine the association of mitoribosomal complexes with mitochondrial membrane
ZELLNER, Laura
The assembly pathways of mitoribosomes, descendants of bacterial ancestors producing proteins encoded by vestigial mitochondrial genomes, remains largely unknown. To shed light on structural features of a precursor of the highly divergent small mitoribosomal subunits (mtSSU) from Trypanosoma brucei, called mtSSU assemblosome, recently characterized by cryoEM, we raised antibodies against two assembly factors present in the complex and against two subunits of the mature mtSSU. We used the antibodies in pilot experiments to determine whether the assemblosome associates with the inner mitochondrial membrane.
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Metody dekontaminace rekombinantních proteinů od bakteriálního lipopolysacharidu
CHARVÁTOVÁ, Lucie
In this work, three decontamination methods based on two-phase micellar system and affinity chromatography were used to decontaminate recombinant proteins from bacterial lipopolysaccharide and to determine which method is the most effective. The efficiency of this method was measured using various recombinant proteins at several protein concentrations. Three different assays, two chromogenic and one fluorogenic, were used to measure the concentration of endotoxins in samples. The most accurate method for measuring the concentration of endotoxins was determined.
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Preparation of mutant variants of Kingella kingae RtxA cytotoxin for membrane topology research
Lichvárová, Michaela ; Osičková, Adriana (advisor) ; Malý, Petr (referee)
Kingella kingae is a facultative anaerobic, β-hemolytic, gram-negative bacterium. It has been shown, that K. kingae is an important cause of invasive infections in young children, especially between 6 to 36 months of age. The most common diseases caused by K. kingae are septic arthritis, osteomyelitis, bacteremia and infective endocarditis. The key virulence factor of K. kingae is the secreted RtxA toxin, which belongs to the RTX toxins family (Repeats in ToXin). These are divided into two categories, hemolysins and leukotoxins, based on the cellular specificity of their action. The broad specificity of the RtxA toxin indicates that RtxA can be classified as a cytolytic RTX hemolysin. RtxA molecules are inserted into the host cell membrane and form cation-selective membrane pores that trigger cation flux. This disrupts normal cell physiology and eventually leads to cell lysis. The aim of this bachelor thesis was to prepare mutant variants of the K. kingae RtxA cytotoxin with lysine substitutions in the pore-forming domain for future study of the membrane topology of the toxin using biotin binding to the lysine residues. In order to observe the topology of the RtxA toxin in the host cell membrane, the toxin must be able to insert to the cell membrane. Therefore, another objective was to determine...
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Characterisation of recombinant mouse glutamate carboxypeptidase III
Janoušková, Karolína ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Glutamate carboxypeptidase II (GCPII, PSMA, NAALADase) is transmembrane metalopeptidase and due to cleavage of substrates β-citryl-L-glutamate (BCG), N-acetyl-L-aspartyl-L-glutamate (NAAG) and polyglutamylated folates (Pte-Glun) is being studied as potential therapeutic target. Enzymes, which could compensate for enzyme activity and functions of GCPII, are thus relevant targets of enzymology as well. One of GCPII's homologs with similar enzyme activity is mouse glutamate carboxypeptidase III (GCPIII, NAALADase II). Enzymatic cleavage has not been determined using recombinant mouse GCPIII yet. It is important to kinetically characterize mouse GCPIII so that we can compare enzyme activity with human ortolog. Then we can find out whether mouse model is comparable with human. Recombinant mouse GCPIII was kinetically characterized. Kinetic parameters (KM, kcat) for recombinant mouse GCPIII were measured for substrates NAAG and BCG using radioactive assay. Experiments with the substrate Pte-Glu2 were analyzed using HPLC method. Although human GCPIII is more effective than mouse ortolog at clearage of NAAG, both enzymes are comparable during hydrolysis of BCG. Those results can contribute to better understanding of the role of GCPIII in the most commonly used animal model.
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Molecular base of plant HSP90-MT interaction
Benáková, Martina ; Krtková, Jana (advisor) ; Malcová, Ivana (referee)
Microtubules (MTs) are one of the essential cell structure that participate in a number of key events in the plant cells and their properties and functions are influenced and modified by many other proteins. These proteins belong to a group of microtubule- associated proteins (MAPs, microtubule-associated proteins). One of the MAPs, the molecular chaperone Hsp90, examines and fulfills a large number of different functions in the cell. Its colocalization with MTs has been demonstrated previously by Freudenreich and Nick (1998) and Petrášek et al. (1998). However, direct interaction with MTs was described only recently using cosedimentation assay. The specific cytosolic isoform of tobacco Hsp90 bound to MTs was called Hsp90_MT due to its ability to bind MTs. It has been also found that the binding to MTs is independent on the activity of ATP (Krtková et al., 2012). The authors also described a positive effect of Hsp90_MT on MT recovery after their exposure to cold stress. Although MT cytoskeleton dynamics is influenced by a large number of MAPs, it is surprising that the molecular mechanism of MAPs interaction with MTs and their MT-binding domains have not been described yet. Therefore, we decided to determine the tobacco Hsp90_MT MT-binding domain by production of a set of recombinant proteins...
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Purification of recombinant proteins by affinity chromatography
Zemek, Ondřej ; Mácha, Jaroslav (advisor) ; Hrdý, Ivan (referee)
The isolation and purification of recombinant proteins is essential for further study of their structural and functional properties. The affinity chromatography is usually the method of choice for this task. In this paper the most used affinity tags are reviewed for their properties and experience with their application. The tags covered here include CBP, MBP, GST, polyhistidine and polyarginine tags, FLAG-tag, Strep-tag II and SpA. The origin and properties of the tags, their influence on form and localization of fusion protein as well as binding, elution and removal are discussed. Keywords: affinity chromatography, recombinant protein, purification, affinity tag
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