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Heterologous expression and purification of human NADPH: cytochrome P450 oxidoreductase
Kostelanská, Marie ; Černá, Věra (advisor) ; Kavan, Daniel (referee)
NADPH: cytochrome P450 oxidoreductase (POR) is an enzyme that is able to catalyze transfer of electrons from NADPH, via two-flavin cofactors, to various redox partners. Therefore, POR is essential for multiple metabolic processes, including reactions catalyzed by cytochromes P450. Due to all microsomal P450s depending on POR for the supply of electrons, disruption of POR may affect all microsomal P450 enzyme activities. Polymorphisms in human POR have been shown to lead to development phenotypes, the severity of which differs significantly depending on the degree of POR impairment. This thesis is focused on the preparation of POR, which is similar to combinatorial allele carrying two single nucleotide polymorphisms P228L and A503V, functionally not clearly characterized at that time. However, disastrous consequences have currently not been noted. Moreover, the presence of A503V has been confirmed as the most common allele, but there is evidence that A503V influences the activity of some redox partners. In present thesis there were two genes subcloned into expression plasmids pCW. The first of which carries the cDNA encoding the POR and the other carrying cDNA encoding POR with the histidine-tag. Expression of the recombinant POR was carried out in the heterologous bacterial system, using...
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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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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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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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