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Preparation of Oryzasin 1 for protein digestion in hydrogen/deuterium exchange.
Šintáková, Kristýna ; Man, Petr (advisor) ; Jurnečka, David (referee)
Hydrogen/deuterium exchange coupled to mass spectrometry (HXMS) is an increasingly popular technique in structural biology. Its spatial resolution strongly depends on the efficiency of the fragmentation or proteolytic cleavage of the studied protein. Therefore, it is desired to search for new proteases that would not only be able to digest the protein of interest under the HXMS conditions, but also to provide the best possible coverage of the protein sequence. Finding optimal conditions for production of Oryzasin 1 aspartate protease for its potential use in HXMS experiments was done in this thesis. Selected production clones were selected from available plasmids, the identity of the produced protein was verified by peptide mapping, and optimal production conditions were found. Based on these results, large-scale protein production and inclusion body isolation were undertaken. (In Czech) Keywords: Oryzasin 1, proteases, hydrogen/deuterium exchange coupled to mass spectrometry (HXMS)
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Inducible expression systems and their use in the study of parasitic organisms.
Horáčková, Vendula ; Doležal, Pavel (advisor) ; Fišer, Radovan (referee)
1 Abstract Inducible expression systems are systems with ability to switch expression of genes of interest on and off. Therefore, they are useful molecular tools for analysis of gene function. Nowadays, there are tens of various inducible expression systems available that differ from each other in level of regulation of gene expression, time of induction, possibilities of use, etc. This work is focused on three of them to illustrate common features of the inducible expression systems which regulate gene expression at the level of transcription. Firstly, systems based on regulation of lactose operon of Escherichia coli are mentioned. Secondly, systems which use regulatory elements of tetracycline resistance-encoding transposon Tn10 of E. coli are described. Third chapter is focused on systems regulated by agonists of ecdysone receptor. In the last chapter cases of use of inducible expression systems in the study of parasitic organisms are summarized.
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The analysis of structural details of the NMDA receptor
Radilová, Kateřina ; Balík, Aleš (advisor) ; Jakubík, Jan (referee)
NMDA receptor is necessary for excitatory transmission in the central nervous system. Altered funtion of the NMDA receptors is associated with many neurodegenerative and neuropsychiatric diseases. All available crystal structures of the NMDAR meant great shift towards our understanding of details of the receptor and its function. Unfortunately, these up- to-date available structures present only certain functional states of receptors and also a few structural data are still missing. For complete comprehension of the process of activation and deactivation of NMDA receptors, we need to supplement the current information with more data. The aim of this thesis was to employ a combination of different approaches (computational modelling, cloning, biochemistry, protein expression and purification and mass spectrometry) to obtain new structural data, by which we would be able to fill in the gaps in current receptor models, especially at various functional states of the receptor. Key words: NMDA receptor, glutamate receptor, computational modelling, structure, cloning, protein expression
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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Study of molecular organization of cytochrome P450 system using photoactivatable proteins
Dědič, Jan ; Hodek, Petr (advisor) ; Novák, Petr (referee)
The cytochrome P450 system plays an important role in metabolism of endogenous compounds and xenobiotics. This system consists of cytochrome P450, NADPH:cytochrome P450 oxidoreductase (CPR), cytochrome b5 and NADH:cytochrome b5 reductase (CYB5R3). Explanation of protein-protein interactions among these reaction partners is essential for understanding the function of the entire system. Covalent cross-linking is a favorable method for studying these interactions. In this work a photo-activatable analogue of amino acid L-methionine (L-photo-methionine) was used as a cross-linking agent. This work is focused on the organic synthesis of L-photo-methionine, expression and isolation of CPR and CYB5R3 as photoactivable proteins containing incorporated L-photo-methionine. Auxotrophic strain of E.coli B834 (DE3) and minimal media were used for the expression. CYB5R3 with incorporated L-photo-methionine was successfully expressed and isolated. The extent of L-photo-methionine incorporation was verified by mass spectrometry. Furthermore, the photo-initiated cross-linking of CYB5R3 with cytochrome b5 was tested. Key words: photolabile amino acid, protein expression, synthesis
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Preparation and characterization of synthetic mRNA coding for pancreatic transcription factors
Loukotová, Šárka ; Hodek, Petr (advisor) ; Jirák, Daniel (referee)
Diabetes mellitus type I is severe autoimmune disease which is caused by destruction of insulin-producing β-cells in pancreas. Diabetic patients are dependent on external usage of insulin during their whole life. Nowadays the only treatment of diabetes type I is transplantation of entire pancreas or isolated Langerhans islets. Due to the fact that this kind of treatment is very demanding and limited availability of suitable donors, the researchers are intensively working on development of new alternative ways how to produce the insulin-producing cells. One of the possible approaches on producing insulin-positive cells is transdifferentiation of pancreatic exocrine cells via transcription factors. In this diploma thesis, the transdifferentiation of exocrine cells AR42J was carried out with in vitro synthesized mRNA encoding transcription factors Pdx1, Ngn3 and MafA. The primary mRNA structure was optimized in order to prepare highly stable mRNA which is correctly translated into the protein. The main stabilizing elements in mRNA structure include 3' and 5' untranslated region derived from highly stable β-globin mRNA. In order to verify the function of synthetic mRNA the immunofluorescence staining of transcription factors has been investigated. Synthetic mRNAs encoding transcription factors Pdx1,...
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Co-operativity of cytochrome P450 system and its impact on drug and carcinogen metabolism
Holý, Petr ; Hodek, Petr (advisor) ; Chmelík, Josef (referee)
The system of mixed-function oxidases (MFO system) has a significant role in metabolism of many endogenous compounds, as well as xenobiotics (for ex. karcinogens, drugs). Membrane-bound haemoproteins called cytochromes P450 are a vital part of that system. Reactions catalyzed by cytochromes P450 are influenced by another protein of the MFO system, cytochrome b5. The mechanism of this cyt b5 agency has not yet been fully described. One of methods used for study of this protein-protein interaction is covalent cross- linking. By replacing one of three methionines in the cyt b5 structure by a photo-reactive analogue (photo-methionine), an analogue of cyt b5 (photo-cyt b5) can be obtained. When activated by UV radiation, the protein covalently bonds cytochrome P450 in a membrane environment. This paper focuses on expression and isolation of a recombinant cyt b5 analogue with only one methionine position (96) in the protein structure and substitution by photo-methionine. Protein was purified in a yiedl of 6 mg from 1 liter of baterial suspension. Analysis by mass spectrometry (MALDI-TOF/TOF) showed methionine to have been substituted by the photo-reactive analogue in approx. 30 %. Photo-cyt b5 was used to fixate transient protein-protein interactions with cytochrome P450 2B4 (CYP2B4). Photo-cyt b5 was...
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Optimalization of expression of photoactivatable cytochrome P450 as a nano-probe for the membrane topology studies of enzymes metabolizing drugs and carcinogens
Smolová, Jana ; Hodek, Petr (advisor) ; Černá, Věra (referee)
The cytochromes P450 are among the most important enzymes involved in the biotransformation of xenobiotics in the body. They are part of the moooxygenase system that interact with other enzymes - NADPH:cytochrome P450 reductase and cytochrome b5. Mutual interaction of enzymes in mooxygenase system are not completely solved. Covalent crosslinking technique could contribute to clarify the possible protein-protein interactions and their consequences. One of the possible realization of this plan is to use photoactivatable cytochrome P450, which after exposure to UV radiation created covalent complex with components of monooxygenase system, with which it is in contact. Therefore, this paper focuses on developing optimal conditions for the production of recombinant cytochrome P450 2B4 in order to gain knowledge for the production of photoactivatable cytochrome P450 with incorporated amino acids L-photomethionine and L-photoleucine. In experiments was cytochrome P450 2B4 expressed in two strains of Escherichia coli, C41 (DE3) and BL21 (DE3) Gold, and two culture flasks, glass Erlenmeyer flask and plastic Fernbach flask. During expression optical density of the bacterial suspension (absorbation at 600 nm) and concentration of cytochrome P450 were measured. Methodology of measuring the concentration of...
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Heterologous expression and purification of human cytochrome b5
Kostelanská, Marie ; Černá, Věra (advisor) ; Bořek Dohalská, Lucie (referee)
The metabolism of xenobiotics and endogenous substances is mediated by a mixed function oxidase system which includes cytochrome b5 participating in catalytic activities of CYP. The mechanism of action of the cytochrome b5 has not been fully elucidated yet. But it is assumed that cytochrome b5 is involved either in direct electron transfer within the mixed function oxidase system or in induction of conformational changes in CYPs. So it is important to gain the pure form of apo-cytochrome b5, devoid of heme, which is not capable of electron transfer and further study the effect of this form on CYP-catalyzed reactions. The obtained results can contribute to understanding the mechanism of cytochrome b5 effects. The transformation of bacterial cells of Escherichia coli BL-21 (DE3) Gold was performed by expression vector pET22b which contained genes for microsomal and erythrocyte cytochrome b5. In order to produce a high level of apoprotein form, the heterologous expression of cytochrome b5 was induced by addition of higher amount of IPTG. Expression was performed at 37řC. This bachelor thesis is primarily engaged in purification of both microsomal and erythrocyte form of cytotochrom b5, especially in its apo-form. However, the productions of holo-cytochrome b5 form always occur in a greater or lesser...
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