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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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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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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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P53 protein isoforms production and purification in the bacterial expression system
Vadovičová, Natália ; Obruča, Stanislav (referee) ; Brázda, Václav (advisor)
Apart from the p53 protein, the TP53 tumor-suppressor gene is expressed as another eleven protein isoforms with the use of alternative splicing, alternative promotors and alternative translational initiation sites. Abnormal expression of these isoforms has been observed in tumor tissues. The binding properties as well as the biological functions are also modulated, due to sequential and therefore structural differences from the p53 protein. p53 is regulated by these isoforms in both suppressive and supportive manner. Explanation of the p53 isoform regulation mechanism in cells could lead to desired alternative splicing of the chosen isoforms, and modulation of isoform expression could be used in cancer treatment based on p53 therapy. Basic information about p53 protein is summarised in the theoretical part of this master thesis, supplemented with recent advances in the field of p53 isoforms, as well as the Gateway cloning method. The main goal of the experimental part was p53 isoform production in a bacterial expression system. Prior to the protein production, DNA sequences coding twelve p53 isoforms were prepared using PCR and Gateway cloning. In total, twelve entry clones and eight expression clones were prepared by cloning the isoforms’ sequences. After the protein production and purification, the detection using SDS-PAGE and Western Blotting was performed with five p53 protein isoforms: p53, 40p53, 40p53 and 40p53. DNA binding properties of p53 protein isoforms will be tested in subsequent research.
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