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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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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 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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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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