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Recombinant expression of immunoglobulin superfamily receptor NKp30
Tůmová, Lucie ; Kavan, Daniel (advisor) ; Šulc, Miroslav (referee)
1 NK cells belong to lymphocytes, which are able to kill some tumor cells and virus-infected cells, produce cytokines and participate in the formation of the adaptive immune response. This bachelor thesis deals with human receptor of NK cells, specifically NKp30. This protein belongs to the group of activating receptors which can activate the cytotoxic function of NK cells. Its three-dimensional structure and the interaction between NKp30 and its physiological ligand B7-H6 has been recently resolved. Howewer the interaction with the other ligands, BAT3 and pp65, remains unknown. The aim of the work was optimalization of expression of the NKp30 receptor extracellular domain in prokaryotic expression system. The structural studies with ligands of this protein may provide further details on the interaction between the receptor NKp30 and ligands BAT3 and pp65. (In Czech)
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Preparation of expression construct of NKp44 receptor
Hajná, Vladimíra ; Kavan, Daniel (advisor) ; Ptáčková, Renata (referee)
NK cells are part of the innate immune system. Their immediate response to pathogen plays a role mainly in the elimination of tumor or virus-infected cells. Recently, NK cells, especially their receptors, are increasingly the subject of various studies. One group of NK cell receptors are known as natural cytotoxicity receptors (NCRs). Nowadays three receptors are belonging to this group, one of them is NKp44. NCR receptors play important role in the activation of NK cells. NKp44 is considered as the first specific receptor for activated NK cells. Its ligands are not yet fully known. Recent research has highlighted one of the potential ligands, the protein PCNA, which surprisingly causes inhibition of NK cell function. The aim of this thesis was to prepare plasmid encoding the extracellular part of the human receptor NKp44. DNA insert was obtained by RT-PCR reaction from isolated NK- 92MI cell line RNA. Subsequently it was ligated into the cloning vector pBSSK(+) and then into production pET30-a(+) vector, resulting in the plasmid for expression of the extacellular portion of NKp44.
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Effect of binding of a fluorescent label on the protein structure and function
Petrovová, Gabriela ; Kavan, Daniel (advisor) ; Martínek, Václav (referee)
Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...
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Structural study of the DCL-1 receptor using mass spectrometry
Růžičková, Barbora ; Kavan, Daniel (advisor) ; Mrázek, Hynek (referee)
DCL-1 (CD302) is a type I transmembrane C-type lectin receptor, which is expressed on monocytes, macrophages, granulocytes and dendritic cells. However, its extracellular domain lacks the amino acids motives essential for carbohydrate binding in the presence of calcium ions, suggesting that it does not have the classic binding capacity found in other C-type lectin receptors such as the mannose receptor. No exogenous or endogenous ligands have been identified yet, though. Due to internal colocalization with F-actin we can assume, that this unconventional lectin receptor plays a role not only in endocytosis and phagocytosis but also in the cell adhesion and migration. The receptor DCL-1 was first identified as a genetic fusion partner of human DEC-205 multilectin receptor in Hodgkin's lymphoma cell lines. The experimental part of this thesis deals with the characterization of disulfide bonds and data acquisition for validation of DCL-1 crystal structure. First the production and refolding conditions were optimized to obtain the highest amount of DCL-1 protein, precisely its extracellular domain. These optimal conditions were used to prepare the protein for in-gel digestion using specific endopeptidases in the presence of cystamine followed by LC-MS analysis. DCL-1 disulfide bonds were determined by comparing...
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Expression and purification of N-terminal fragment of filamentous hemagglutinin from Bordetella Pertussis in E. Coli
Jurnečka, David ; Stiborová, Marie (advisor) ; Kavan, Daniel (referee)
: Whooping cough is highly contagious disease caused by gram-negative bacteria Bordetella pertussis. During infection the bacteria produces many types of toxins and adhesive molecules including a filamentous hemagglutinin(FHA). FHA is 220 kDa surface-exposed and secreted protein, which plays a key role in host-cell interactions. The project aims at construction of heterologous expression system for production of the N-terminal part of B. pertussis FHA (FHA1-862) in E. coli. The expression vector is composed of system of two independent T7/Lac promoters and enables secretion of FHA1-862 into the culture media. Downstream of the first promoter is fhaB gene encoding FHA1-862 and the letter is followed by fhaC gen encoding the FhaC transport protein, which allows translocation of FHA from periplasmic space to extracellular milieu. FHA1-862 was successfully secreted in E. coli strain BL21 carrying plasmid pMM100 (Laclq) at 30 řC and purified by affinity chromatography on Cellufine resin. These results indicate that FHA1-862 protein can be produced in E. coli, however, the system is inefficient and the yield of the protein is very low. (In Czech)
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Recombinant production of bovine NK cell receptors
Böerová, Nikola ; Kavan, Daniel (advisor) ; Šulc, Miroslav (referee)
NK cells, which are part of the innate immune system, are increasingly gaining attention, especially due to their cytotoxic ability to kill tumor cells of certain lines and certain viral, bacterial or parasitic infestation of the body. They lay a role in organ transplantation, the fight against HIV and other autoimmune diseases. NK cells have been studied since the 70th of the 20th century, but the structures and physiological ligands of their receptors remain only partially understood, as does the exact role of these cells in the organism. They communicate with others through their receptors, that recognize the lack of expression of MHC class I glycoproteins on the surface of target cells, thereby preventing cell recognition by cytotoxic lymphocytes. This diploma thesis deals with the research of receptors from cattle (Bos taurus), which is not a traditional laboratory animal and my task was to contribute to research of the structure of this group of proteins. I dealt with the recombinant production of some of the most important representatives of NK receptors CD69, NKRP1 and NKG2D in bacterial cells. The findings published in this thesis are a continuation of my bachelor thesis and together can be beneficial for further research into structural proteins and thus may help as in veterinary medicine...
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Studies on DCL-1, receptor of dendritic cells, using NMR techniques
Pospíšilová, Eliška ; Kavan, Daniel (advisor) ; Schneider, Bohdan (referee)
(EN) The DCL-1 receptor (CD302) is predominantly expressed on the surface of dendritic cells and according to its sequence similarity DCL-1 is classified as a C-type lectin. Since its extracellular domain lacks single motives for carbohydrate binding in coordination with calcium ions, it is probable that the process of carbohydrate binding does not occur through the classical pathway as described in the case of mannose receptor or the DEC-205 receptor. Due to its colocalization with F-actin there is a presumption, that DCL-1 plays a role in cell adhesion and migration. Another role of DCL-1 could be the participation in endocytosis and subsequent targeting to lysosomes. DCL-1 was also put in connections with various pathologies in last few years Experiments described in this work can be divided into three sections. In the first part I dealt with production of a protein construct based on the extracellular domain of DCL-1. The protein was produced in M9 minimal medium with the only source of nitrogen 15 NH4Cl and the only source of carbon 13 C glucose. The result was 15 N,13 C labelled protein, used for NMR measurements. The second part is dedicated to the analysis of NMR spectra, which enabled us to assign frequencies of protein backbone and aliphatic side chains atoms. On the base of the backbone...
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Sulphated metabolites of silybin and their interactions with sulphatases
Ježková, Zuzana ; Weignerová, Lenka (advisor) ; Kavan, Daniel (referee)
This bachelor thesis solves the question of sulphated metabolites of silybin by their interactions with sulfatases. Silybin is flavonolignan prepared from the seeds of milk thistle (Silybum marianum). Sulfate of silybin (metabolite) has not been isolated from organism so far thus its exact structure has not been determined. In this work synthesis of silybin-7,23-disulfate from natural silybin is presented. Silybin disulfate was isolated and spectrally characterized (NMR, MS). In the next step the kinetics of the reaction pNPS with sulfatase from Helix pomatia was measured: Km = 0.0494 mmol/l a Vmax = 0.0325 mmol/dm3 /min. Conditions for sulfatase reaction from Helix pomatia were selected according to literature. One of the main goal was to determine, whether silybin-7,23-disulfate is a substrate and/or an inhibitor of sulfatase from Helix pomatia. Activity measurements of the sulfatase from Helix pomatia in the presence of various concentrations of silybin-7,23-disulfate enabled us to determine that silybin disulfate at concentration higher than 0.2 mM is a strong inhibitor of the sulfatase tested. Series of reactions with sulfatase from Helix pomatia were performed to determine whether silybin-7,23-disulfate is a substrate. Reactions were monitored by HPLC and it was demonstrated that the...
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Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry
Kavan, Daniel
CD69 is considered as the marker receptor of activated lymphocytes and is expressed at sites of active immune response. Physiologically it appears in the form of covalently bound homodimer, however after examining its three- dimensional structure we suggested Q93 on one subunit and D88 with E87 on the other one to participate on the inter-subunit interactions. Even more profound intertwining was observed in case of R134 of one subunit with A136 and Y135 on the other one. Therefore Q93, R134 or both were mutated into alanines and showed the monomeric form just in case of double-mutant. This fact influenced significantly also the binding of ligands. While the Kd values for binding of GlcNAc was approximately 10-5 M in case of monomeric form, in case of dimeric form it was 10 times lower and even 100 times lower in case of the longest covalently bound dimers. Although the gel filtration retention time decrease was observed, which could indicate a change in molecular fold, the value of experimentally determined sedimentation coefficient was identical. Moreover neither the comparison of HSQC NMR spectra before and after ligand saturation revealed any significant shifts. Hydrogen deuterium exchange is a chemical process in which a covalently bonded hydrogen atom is replaced by deuterium or vice versa. As...
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Studies of NK cell receptors and other proteins using recombinant expression and mass spectrometry
Kavan, Daniel ; Bezouška, Karel (advisor) ; Tučková, Ludmila (referee) ; Řehulka, Pavel (referee)
Charles University in Prague Faculty of Science Department of Biochemistry Studies of NK cell receptors and other proteins using recombinant expressions and mass spectrometry Summary of Ph. D. Thesis Daniel Kavan Supervisor: Prof. RNDr. Karel Bezouška, DSc. Prague 2010 Daniel Kavan Introduction Introduction NK cells and CD69 as one of their surface receptors Natural killer cells (NK cells) are the subpopulation of large granular lymfocytes, which lacks the surface receptors typical for B cells or T cells. They are characterized by the presence of NKp46 and NKp30 [Moretta L. et. al. 2002], however. They were named natural killers according to their function in the organism, as they do not need any activation and nevertheless they are able to eliminate abnormal (i. e. infected or transformed) cells from the tissue [Kiessling R. et. al. 1975]. This function is dependent on scanning the major histocompatibility complex (MHC) class I molecules of ambient cells. The resulting action (killing or not killing the target cell) is dependent on the balance of activating and inhibiting signals mediated by the NK cell surface receptors and forwarded to the specific signaling pathway [Raulet D. H. et. al. 2001]. Specificity of NK cells is not based only on one type of antigen receptor as it is in case of T and B cells,...
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