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Preparation and characterization of a bacterial protein YddV, a globin-coupled oxygen sensor diguanylate cyclase
Křížová, Věra ; Martínková, Markéta (advisor) ; Man, Petr (referee)
Heme-containing proteins are very important for proper function of various organisms, both prokaryotic and eukaryotic. The most well-known ones are myoglobin, hemoglobin and various peroxidases and cytochromes. The group of heme-containing proteins was recently extended by a new type of proteins, which are called heme-containing sensor proteins. These proteins consist of two domains, sensor domain, which is able to detect a signal (either a molecule of heme or a molecule of gas), and functional domain, which is often an enzyme or a transcriptional factor. Activity of the functional domain depends on the concentration of signal molecule, which binds to the sensor domain, which then causes change of its structure. Consequently, change of structure of the sensor domain also leads to change of structure of the functional domain. Heme-containing sensor proteins can be further divided into two smaller groups: heme sensor proteins and gas sensor proteins. Heme sensor proteins share a lot of features, for example binding of heme to a thiolate residue from cysteine, CP motif, etc. Gas sensor proteins detect a molecule of gas, especially oxygen, carbon monoxide and nitrogen monoxide. There have already been conducted various studies focusing on heme-containing sensor proteins, however, the mechanism of signal...
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Important roles of heme as a signal and a gas-sensing site: heme-sensing and gas-sensing proteins
Fojtíková, Veronika ; Martínková, Markéta (advisor) ; Hudeček, Jiří (referee)
Heme-containing sensor proteins are heme proteins, which are divided into two groups: heme-sensing and gas-sensing proteins. The function of heme-sensing proteins is affected by heme availability. Association (or dissociation) of heme moiety of heme-sensing protein regulates various physiological functions, including protein kinase activity, transcription and other important functions essential for cell survival. In gas-sensing proteins, heme acts as the sensing site for binding of gaseous molecules (including O2, NO and CO) and indirectly regulates physiological functions, including protein kinase activity, transcription and other important functions essential for cell survival. The recent studies on heme-containing senzor proteins published in scientific journals are summarized in this thesis. The experimental part of this thesis focused on the specific heme-containing sensor protein - a globin-coupled histidine kinase from Anaeromyxobacter sp. strain Fw 109-5 (AfGcHK). The aim of this thesis was to amplified and isolate plasmid carrying gen for AfGcHK. Consequently the protein was expressed in E.coli BL-21(DE3) and the protein was isolated. Based on the results, the isolation process was optimized. Moreover, the purified preparation of isolated AfGcHK was prepared in more than 99% of homogeneity....
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Růst Mycobacterium smegmatis na agarovém médiu a agarovém médiu pokrytém celofánovou folií - morfologická a proteomová studie
Ramaniuk, Volha ; Weiser, Jaroslav (advisor) ; Beranová, Jana (referee)
Biofilm formation is one of the most common bacterial survival strategies. Majority of bacterial species are able to form these three-dimensional structures, including pathogens like Mycobacterium tuberculosis. Representatives of Mycobacterium genus widely occur in the nature, although they can cause serious problems when they appear in medical equipment and artificial replacements of the human body. Non-pathogenic Mycobacterium smegmatis mc2 155 was used as a model organism in our experiments. We investigated morphology of the three- and six-day-old colonies (in fact biofilms) on agar and agar covered with cellophane using Stereo microscope and Scanning Electron Microscope. We found that a type of surface as well as a carbon source has a great influence on the morphology of the M. smegmatis colonies. We isolated proteomes from the agar and cellophane cultures and from planktonic culture. Two-dimensional electrophoresis was used as the main proteomic method. Proteomic data were analyzed using PDQuest software. Then the sets of proteins detected by qualitative and quantitative analyses were compared using Venn diagrams. As a result, we recognized 7 unique proteins that might be specific for recognition and adhesion of bacteria to the cellophane, no unique protein in agar proteome and 46 unique...
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Study of modified amino acid incorporation into cytochrome b5
Koberová, Monika ; Hodek, Petr (advisor) ; Pavlásková, Kateřina (referee)
A cytochrome b5 (cyt b5) can influence cytochrome P450 (CYP)-dependent reactions. In consequence of these reactions cytochrome b5 can participate in substance activation (for example drugs, carcinogens) or it can influence proportions of formed metabolites. A mechanism of cyt b5 action has not been fully explained yet. Elucidation of protein-protein interactions in monooxygenase system could explain of the mechanism of cyt b5 action. To study these interactions by using cross-linking techniques is necessary to prepare photolabile cyt b5, which after photoactivation generated higly reactive intermediates which can create a complex with nearby components of the monoogynesase system. This thesis describes how was developed the method for the production of recombinant cyt b5 with modified amino acids. Cyt b5 was expressed in a bacterial strain E. coli BL-21 (DE3) Gold. Before the expression induction, cells were transformed into the limiting medium (DMEM-LM) which did not contain L-leucine and L-methionine. The limiting medium was supplemented by deuterated amino acid d3-methyl-L-methionine and D,L-Leucine. Expressed cyt b5 was isolated and incorporation of d3-methyl-L-methionene has been verified by mass spectrometry. Cyt b5 was obtained mainly as the apoprotein (apo-cyt b5). That is why in this...
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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