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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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Structural analysis of the interaction interface between an antibody and a protein epitope
Moravec, Jan ; Krejčíř, Radovan (referee) ; Müller,, Petr (advisor)
The diploma thesis deals with the study of the mouse monoclonal antibody EEV1-2.1, which was created in the RECAMO laboratories by immunization of mice with the Hsp90 protein and subsequent fusion of splenocytes with a mouse myeloma line. The theoretical part of this work focuses mainly on the study of antibodies, especially their structure, genetics and the description of monoclonal antibodies. In this part of the work, you can also find a cross-section of modern biotechnological trends that are currently used for the production of antibodies. CHO cells and tetracycline inducible systems are also described. The aim of the thesis was to characterize the mentioned antibody. Using bioinformatics methods and prediction software to predict and describe its structure, especially then to analyze hypervariable CDR regions. Another goal was to clone the light and heavy chains of the antibody using the Gateway method. The final step was the creation of a tetracycline inducible system for efficient production of the antibody itself. The experimental part of the work initially deals with the isolation of the RNA sequence of the antibody using commercial kits. Next, the method of amplification of the given sequence using PCR with reverse transcription is described. The following is a description of the Gateway cloning method, which allows convenient insertion of the selected gene into the target vector. The basic bioinformatics methods used to study the structure of antibodies are also explained, as well as the prediction of a more complex 3D structure of the antibody, including possible interactions with the HSP90 protein. This section describes the methods of transfection of ExpiCHO cells and inducible antibody production using the tetracycline inducible system. The thesis led to the successful cloning of the light and heavy chain of the antibody and the subsequent insertion of the expression vector into the production ExpiCHO cells. The expression system based on induction by the addition of doxycycline was then successfully tested by several methods and the results show that the inducible system is not only functional, but can even lead to increased production of monoclonal antibody compared to conventional methods.
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Diversity of Polycomb complexes and their function
ŘÍHA, Luboš
The aim of this bachelor thesis is testing and further development of a vector system that should help clarify the alleged functional redundancy of Polycomb repressive complex 2 (PRC2) subunits. The theoretical part introduces the field of epigenetics and the role of Polycomb group complexes (PcGs) in Arabidopsis thaliana (mouse-ear cress) is explained. The issue of PRC2 subunits SWN and CLF redundancy is set in context and the tested hypothesis is explained. Genetic engineering tools relevant to this study are presented. Finally, the background of the promoter and marker vectors developed in the practical part is explained. In the practical part vectors with markers and promoters are developed and transgenic plants were grown on selection and genotyped. Results are presented and discussed.
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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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