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Study of oligandrin protein secreted by oomycete Pythium oligandrum
Neykulova, Anastasia ; Bělonožníková, Kateřina (advisor) ; Adámková, Lyubina (referee)
Oomycete Pythium oligandrum acts as a mycoparasite of pathogenic fungi, bacteria and oomycetes in soil due to the production of a number of hydrolytic enzymes for the degradation of polysaccharides and proteins in the cell wall of the host. At the same time, P. oligandrum can interact with plant roots through specific elicitors and stimulate plant defense and growth. Thus, P. oligandrum is a successfully used environmentally friendly biological control agent of plants. Among the secreted elicitors P. oligandrum to the low molecular weight proteins belong oligandrins (~10 kDa), which have a characteristic structure and a conserved sequence among the group of so-called elicitins, and at the same time are not homologous to plant proteins. In the framework of this bachelor thesis, was analyzed the production of oligandrin in various types of growing media after cultivation of P. oligandrum. The total proteolytic activity and the content of phenolic substances as other possible elicitors of plant defense reactions were also observed in culture media. Further, this work focused on the possibility of recombinant oligandrin expression in E. coli and its subsequent purification. Key words: Pythium oligandrum, oligandrin, elicitors, cultivation, recombinant expression [IN CZECH]
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Preparation of growth factor TGF-β3 with coiled-coil tag
Otépka, Tomáš ; Vaněk, Ondřej (advisor) ; Kubíčková, Božena (referee)
Growth factors represent a group of significant substances in the metabolism of organisms. They are signaling molecules that control cell activity at the endocrine, paracrine, or autocrine levels. They act as key mediators binding to cell receptors, triggering a cascade of reactions leading to the regulation of genetic transcription in the cell nucleus and stimulation of cellular response. Growth factors influence various physiological functions such as cell proliferation, cell differentiation, and tissue healing. The utilization of growth factors is evident, for example, in regenerative medicine. For a similar purpose, research has been initiated to prepare the growth factor TGF-β3 with the possibility of attaching it to a polymeric carrier using a coiled-coil tag. This work focuses on the recombinant production of TGF-β3 - its analog with a latency-associated peptide (LAP) - and the application of techniques applicable to the intention of this work, specifically, attaching the protein to a polymeric carrier based on amino acids. Given the structural complexity with which growth factors are physiologically released from cells, the preparation of growth factors with a coiled-coil tag in vitro represents an unexplored challenge in the field of recombinant protein expression. In our HEK293T cell line...
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Structural and functional characterization of inhibition of a coronaviral methyltransferase.
Ivanovská, Dana ; Bouřa, Evžen (advisor) ; Faltová, Lenka (referee)
Coronaviral methyltransferases participate in the modification of the 5'-end of viral RNA. Their enzymatic activity not only ensures efficient mRNA translation, but also allows the virus to escape the recognition of the innate immune system. This work is focused on the SARS-CoV- 2 methyltransferases (the methyltransferase domain of the nonstructural protein 14, MT14, the nonstructural protein 16 and its cofactor - the nonstructural protein 10, nsp16/10), which represent attractive molecular targets for therapeutic intervention. The aim of this work was to structurally characterize the coronaviral methyltransferases in complex with various small molecules. The recombinantly prepared proteins were purified and subsequently subjected to crystallization trials. The obtained crystals of the nsp16/10 heterodimer in complex with sinefungin were soaked in a solution containing a S-adenosyl-L-homocysteine analogue. Crystals suitable for X-ray crystallography of MT14 in complex with two different inhibitors were obtained by optimizing the identified primary crystallization conditions. The aquired structural data of the MT14 inhibitory complexes will serve as a basis for the design of new small molecule inhibitors targeting the S-adenosyl-L-methionine binding site. Keywords: methyltransferase, nsp14, nsp16,...
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Production of cysteine cathepsins and structural characterization of their interaction with peptidomimetic inhibitors
Wichterle, Filip ; Mareš, Michael (advisor) ; Knejzlík, Zdeněk (referee)
Cysteine cathepsins participate in many pathological processes such as cancer and neurodegen- erative, cardiovascular, or autoimmune diseases. This work is focused on cathepsins B, L, and V (CatB, CatL, CatV), which represent attractive targets for the development of inhibitors as potential chemotherapeutics and diagnostic tools. The aim of this study was to prepare these cathepsins and structurally characterize their complexes with selected synthetic peptidomimetic inhibitors. Recombinant CatB and CatL were prepared in the yeast Pichia pastoris, and the expression conditions were optimized for the production of cathepsin zymogens. A chromato- graphic purification protocol was designed for CatB and CatL, while CatV was purified using a previously developed procedure. The obtained enzymes were used to prepare complexes with six peptidomimetic inhibitors equipped with a carbamate, vinyl sulfone, or azanitrile warhead, which selectively target CatB, CatL, and CatV, respectively. Their inhibition parameters were determined in a kinetic assay and initial crystallization conditions were identified. After opti- mizing the crystallization conditions for CatB with three carbamate inhibitors, crystals suitable for X-ray crystallography were obtained. Based on the crystal structures of these complexes, the...
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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Preparation and use of acid proteases for digestion in H/D exchange.
Kukla, Jan ; Man, Petr (advisor) ; Pompach, Petr (referee)
- 5 - Abstract Hydrogen/deuterium exchange coupled to mass spectrometry (HX-MS) utilizes the spontaneous exchange of protein backbone amide hydrogens for deuterium atoms from solution to gain information about changes in protein structure. To localize these changes to specific areas of the protein, enzymatic digestion by aspartate proteases is used. The proteases' ability to produce small overlapping peptides and to provide full sequence coverage of the studied protein is essential for pinpointing the protein regions of interest. In this study recombinant proteases nepenthesin I (Nepenthes gracilis) and rhizopuspepsin (Rhizopus chinensis) were prepared and compared to commercially available proteases porcine pepsin A and aspergillopepsin (Aspergillus saitoi). The comparison was performed using various activity assays, where the effects of pH, temperature and denaturing and reducing agents on the activity of the proteases were studied. All four proteases were also immobilized on a polymeric resin POROS and their activity in an online HX-MS digestion setup was tested using myoglobin as a model substrate.
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Recombinant expression of transcription factor TEAD1.
Řeháková, Jana ; Novák, Petr (advisor) ; Bláha, Jan (referee)
TEAD1 is one of the members of the TEAD transcription factor family. This transcription factor is very important, for example for development of organs. The structure of the entire TEAD1 transcription factor is not now yet known. Nevertheless, structure of two important domains are known. The TEA binding domain, which is important for the binding of the transcription factor TEAD1 to DNA, and the transactivation domain, to which coactivators bind. TEAD1 binds to DNA and upon coactivator bind it affects the transcription of various genes. Genes, that are affected by the transcription factor TEAD1 includes genes regulating proliferation, differentiation and apoptosis of cells. TEAD1 is also the target of the Hippo signalling pathway, which is active in adulthood and prevents abnormal growth of organs. Important for the activity of transcriptional factor TEAD1 are post-translation modifications, such as palmitoylation and phosphorylation. To discover the entire structure of the transcriptional factor TEAD1 and the way it interacts with DNA, the transcriptional factor TEAD1 was prepared recombinationally by expression in cells of Escherichia coli bacteria. Suitable conditions for production of the transcriptional factor TEAD1 were found and the cleavage of the histidine tag by thrombin was performed....
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Recombinant expression of RhoA protein with affinity tag.
Filipová, Barbora ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
The rate of intestinal infections associated with antibiotics has been rising steeply. Virulent toxins of C. difficile, Yersinia and many other bacteria target the RhoA protein. The protein is a GTPase involved in signalling pathways, its major function being the regulation of the actin cytoskeleton. The above mentioned toxins render this protein non- functional, which may lead up to a fatal destruction of the cell. The present diagnostic methods of these infections are insufficient. The use of protein chips and mass spectrometry to detect any RhoA protein modifications seems to provide a feasible method of determining these intestinal bacterial diseases. Therefore the purpose of this study is to use recombinant methods to prepare the desired RhoA protein with a bound affinity probe which will serve to its immobilisation on a biochip. The thus prepared protein will be subsequently used to diagnose the previously described diseases. In Czech.
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Preparation and study of human lymphocyte receptor LLT1
Bláha, Jan ; Vaněk, Ondřej (advisor) ; Konvalinka, Jan (referee)
Natural killer (NK) cells are an intensively studied part of immune system, possessing unique ability to recognize and induce death of tumor and virus-infected cells without prior antigen sensitization. Their function is regulated by a fine balance of signals induced by multiple activating and inhibitory cell surface receptors and their interaction with the ligands present on the target cell. Recent research in their C-type lectin-like receptors repertoire has shown that ligands of some of these previously orphan receptors lie within their own family, describing a lectin-lectin interaction. This is the case of human inhibitory receptor NKRP1 (gene KLRB1) and its ligand LLT1 (gene CLEC2D). Previous studies have shown that overproduction of LLT1 in cancer cells or lower production of NKRP1 in NK cells is connected to cancerous manifestations. This master's thesis shows a successful production of the extracellular part of LLT1 utilizing a mammalian expression system based on transient transfection of modified human embryonic kidney (HEK) cell lines. It was found that the five cystein residues contained within the lectin domain of LLT1 tend to cause misfolding and formation of aggregates. Stabilization of the domain was achieved by restoration of the sixth cystein residue at the evolutionary conserved...
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Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography
Vaněk, Ondřej ; Bezouška, Karel (advisor) ; Hrabal, Richard (referee) ; Bařinka, Cyril (referee)
Department of Biochemistry, Faculty of Science, Charles University in Prague 2010 Structure and function of C-type lectin NK cell receptors studied by recombinant expression and protein crystallography Abstract of Ph.D. thesis Ondřej Vaněk Supervisor: Prof. RNDr. Karel Bezouška, DSc. Natural killer cells (NK cells) were found out for their ability to spontaneously kill certain allogeneic tumour cell lines, without any previous sensitization. NK cells are part of non- adaptive immune response with very short reaction time against pathogens such as viruses, intracellular bacteria, parasites, and they are responsible for elimination of certain tumour cells and thus they are able to fight against malignancy and formation of metastasis. Activity of NK cells is regulated by the balance between activation and inhibitory signals mediated by the NK cell surface receptors. From the structural point of view, the majority of NK cell surface receptors could be classified as the C-type lectin or immunoglobulin-like receptors. One of many C-type lectin subgroups are type II lymphocyte receptors that are expressed on the NK cell surface. This study had two main aims. The first one was to find suitable expression and purification systems for selected C-type lectin receptors of NK cells and the other one was to perform their...
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