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Preparation of constructs for protein isolation and its testing
Osadchuk, Olha ; Kostovová, Iveta (referee) ; Brázda, Václav (advisor)
This study is focused on describing of recombinant protein production. Protein p53 was chosen, as one of the most important tumor suppressor proteins, for studying this issue. The p53 protein is responsible for the gene regulation, control of cell cycle and DNA replication. P53 is the most mutated gene in human cancer. Several point mutations of p53 protein was chosen for work with. The theoretical part describes main properties of protein, expression systems, Gateway cloning system and methods of protein purification. In the experimental part are described the procedures of preparing of the expression vectors by Gateway technology, cell transformation and DNA plasmid isolation. Using cloning technology were prepared three expression clones, they were transformed into competent cells and after was done DNA isolation.
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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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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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Substrate cleavage by mammalian Dicer isoforms
Kubíková, Jana ; Svoboda, Petr (advisor) ; Pospíšek, Martin (referee)
Host organisms evolved antiviral responses, which can recognize the viral infection and deal with it. One of the frequent signs of viral infection in a cell is appearance of double-stranded RNA (dsRNA). One of the pathways responding to dsRNA is RNA interference (RNAi), which functions as the key antiviral defence system in invertebrates and plants. Mammals, however, utilize for antiviral defence a different dsRNA-sensing pathway called the interferon response. RNAi functions only in mammalian oocytes and early embryonal stages although its enzymatic machinery is present in all somatic cells, where it is employed in the microRNA pathway. A previous study indicated that the functionality of RNAi in mouse oocytes functions due to an oocyte-specific isoform of protein Dicer (DicerO ), which is truncated at the N-terminus. In my thesis, I aimed to assess whether DicerO processes RNAi substrates more efficiently in vitro than the full-length Dicer (DicerS ), which is found in somatic cells. Therefore, I developed Dicer purification protocol for obtaining both recombinant mouse Dicer isoforms of high purity. I examined their activity in a non-radioactive cleavage assay using RNA substrates with structural features characteristic of RNAi substrates. My results suggest that recombinant DicerO and DicerS do not...
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Recombinant preparation of TEAD transcription factor.
Lišková, Růžena ; Novák, Petr (advisor) ; Staněk, Ondřej (referee)
Recombinant preparation of TEAD transcription factor (abstract) The TEAD family transcription factors play an important role during devolopment of organisms, where their main purpose is to regulate organ size by activating expression of proteins involved in cell growth and differentiation and apoptosis inhibition. TEAD proteins activity is regulated by signalling pathways and interactions with coactivators. Disregulation of these mechanisms can lead to development of tumors, which is the reason why TEAD proteins became an interesting target for development of new anticancer drugs based on inhibiting their activity. There are several possibilities how to inhibit activity of a transcription factor including blocking its bond to DNA. To design a new drug that blocks transcription factors binding to DNA the structural basis of interaction of these two molecules has to be known first. In this thesis the DNA binding domain of human protein TEAD1 was prepared using the technique of recombinant expression in bacteria E. coli. Suitable conditions of protein production were found and the DNA binding domain of TEAD1 protein was purified so it will be possible to use it for structural analysis of its intraction with DNA.
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Recombinant preparation of DNA binding domain of transcription factor TEAD4
Zákopčaník, Marek ; Novák, Petr (advisor) ; Šulc, Miroslav (referee)
6 Abstract Transcription factors play a key role in the management of cell growth and differ- entiation and their deregulation is associated with many cancers. TEAD proteins utilise highly conserved DNA binding domain to recognise specific DNA sequences. This domain could facilitate new drug design and development. The goal of this master thesis includes recombinant preparation of DNA binding domain of transcriptional factor TEAD4 extended by a part of an unstruc- tured variable sequence, which connects this domain with transactivation domain. Purification steps include affinity chromatography followed by size exclusion chro- matography. The characterization of produced protein was performed by mass spectrometry and finally, native gel electrophoresis was used to prove the ability of the produced protein to bind DNA. During purification steps, a fragmentation from C-terminus was observed. Based on analysis of the mass spectra, three most represented forms of produced protein were described all of which were fragmented. The most abundant form (55%) consisted of amino acids 30-131 from TEAD4 protein. Second most abun- dant form (18%) consisted of amino acids 30-144 and the third form consisted of amino acids 30-81. Native gel electrophoresis verified the ability to bind DNA, the efficiency was however lower...
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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 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 of constructs for protein isolation and its testing
Osadchuk, Olha ; Kostovová, Iveta (referee) ; Brázda, Václav (advisor)
This study is focused on describing of recombinant protein production. Protein p53 was chosen, as one of the most important tumor suppressor proteins, for studying this issue. The p53 protein is responsible for the gene regulation, control of cell cycle and DNA replication. P53 is the most mutated gene in human cancer. Several point mutations of p53 protein was chosen for work with. The theoretical part describes main properties of protein, expression systems, Gateway cloning system and methods of protein purification. In the experimental part are described the procedures of preparing of the expression vectors by Gateway technology, cell transformation and DNA plasmid isolation. Using cloning technology were prepared three expression clones, they were transformed into competent cells and after was done DNA isolation.
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NMR study of the extracellular part of the mouse Nkr-p1b receptor from natural killer cells
Skála, Kristián ; Chmelík, Josef (advisor) ; Martínek, Václav (referee)
Protein Nkr-p1b is a surface receptor of cytotoxic NK cells, that mediates inhibitory signal toward the body's own cells. In this study, the ligand binding domain of the mouse protein receptor Nkr-p1b (mNkr-p1b LBD) was prepared by recombinant expression in E. coli cells. Isolated protein was subsequently used for NMR structural analysis. Prediction of protein secondary structures ratio was carried out using three different methods (CD, PSIPRED and TALOS). Results correlate well with the structure of CTLD domain, that plays a key role in ligand binding and thus to function of Nkr-p1b receptor. We managed to prepare this protein in a form suitable for NMR experiments. Based on the data obtained by NMR spectra analysis, a preliminary model of the mNkr-p1b LBD protein structure was created. However, for more precise learning of the 3D structure accurate positions of individual atoms need to be determined by other NMR spectra evaluation in the next phase. Explaining the structure of the ligand binding domain of mNkr-p1b protein could help to better understand the complex mechanism of activation of NK cell cytotoxic activity, thereby contributing to its controlled use as a therapeutic against some viral and tumor diseases.
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