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Production and characterisation of human C1 inhibitor and Plasmodium falciparum PfMSP3.1 recombinant proteins for structural studies
Čápová, Kateřina ; Konvalinka, Jan (advisor) ; Heidingsfeld, Olga (referee)
PfMSP3.1 is one of the surface proteins of the intracellular parasite Plasmodium falciparum, which causes malaria. As one of the evasion strategies of the immunity system of the human host this protein interacts with one regulator of the complement system - C1 inhibitor. Determining the exact binding site and its structural assessment would help to better understand the interaction between the parasite and the host, which is necessary for the disease progression and thus for the development of a potential therapy. In the theoretical part of the thesis, the life cycle of Plasmodium falciparum, the role of the parasite stage called merozoite, the role of its surface proteins, including merozoite surface protein 3, in the attack of red blood cells by the parasite, are described in more detail. It also briefly describes the complement system, its activation pathways and the regulation of these pathways. The experimental part includes the cloning of plasmids to produce C1 inhibitor and various forms of merozoite surface protein PfMSP3.1, transfection of S2 insect cells with these plasmids, subsequent protein expression in S2 cells and their purification. In the second half of the experimental part, we tried to create complexes of C1 inhibitor with individual PfMSP3.1 forms and an attempt to crystallize...
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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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Analysis of trypsin inhibitor-like cysteine-rich domain-containing peptides (TIL-domain inhibitors) from the tick Ixodes ricinus
PECHOVÁ, Hana
The master thesis deals with the analysis of trypsin inhibitor-like cysteine-rich domain-containing peptides (TIL-domain inhibitors) extracted from salivary glands of a tick Ixodes ricinus. It comprises the initial bioinformatical analysis of TIL-domain containing peptide family from I. ricinus, molecular cloning of the representative protein into expression vector, followed by production of recombinant TIL-domain inhibitor in bacteria. A representative TIL-domain gene is proved for their structure, biochemical properties as well as immunomodulatory and anticoagulation features.
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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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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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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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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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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 the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A
Chvojanová, Zuzana ; Kovařík, Miroslav (advisor) ; Němečková, Ivana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences The University of Alcalá, Faculty of Medicine, Department of biomedicine and biotechnology Student: Zuzana Chvojanová Supervisor: PharmDr. Miroslav Kovařík, Ph.D. Consultant: María Isabel Arenas Jimenéz Title of the diploma thesis: Study of the variations in the expression of different adhesion and cytoskeletal proteins of podocytes (E-Cadherin, Podocin, Vimentin) due to Bisphenol A Bisphenol A (BPA) is one of the most widespread compounds in the world, producing over 6 billion metric tons per year. It is widely used as part of polycarbonate plastics and epoxy resins, from which reusable plastic bottles, food boxes and some medical equipment are made. It is also used to coat the inner layer of the cans. Previous studies have shown that BPA contributes to many chronic diseases in the human body, such as kidney disease - diabetic nephropathy. Podocytes - terminally differentiated cells of the Bowman's capsule in glomerulus - are an integral part of the filtration barrier, where they play an important role in preventing the plasmatic proteins from penetrating to the urine. Therefore, in this study, we looked at the effect of BPA on these cells and their particular proteins, using both in vivo and...
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