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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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Waste materials utilization for preparing hydrolysates for the fermentation phase.
Vadovičová, Natália ; Hrstka, Miroslav (referee) ; Babák, Libor (advisor)
Bachelor thesis focuses on the study and comparison of different types of hydrolysis, their optimization and maximization of yields for the upcoming fermentation. Orange peel was chosen as a substrate to conduct the experiments. First, the substrate was mechanically grinded to form a suspension. Each suspension then underwent one out of the examined methods of hydrolysis. Chosen methods were physical, such as microwaves, increased temperature or ultrasound, and chemical acidic and alkaline. Combinations of both types were also examined. The last optimized method was enzymatic hydrolysis. First set of experiments was conducted using enzymes Novozymes® NS50013 and NS50010. Production of cellulase and pectinase enzymes by A. niger during solid-state fermentation that lasted 10 days was also studied. The yields of reducing sugars of all the experiments were calculated using the Somogyi-Nelson method. Enzymatic hydrolysis was proven to be the most effective using the combination of both of the enzymes for a period of 96 hours at pH = 4.5 and temperature 45 °C. Yield of the reducing sugars under these conditions reached 27,4241 ± 0,0007 gl-1.
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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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Waste materials utilization for preparing hydrolysates for the fermentation phase.
Vadovičová, Natália ; Hrstka, Miroslav (referee) ; Babák, Libor (advisor)
Bachelor thesis focuses on the study and comparison of different types of hydrolysis, their optimization and maximization of yields for the upcoming fermentation. Orange peel was chosen as a substrate to conduct the experiments. First, the substrate was mechanically grinded to form a suspension. Each suspension then underwent one out of the examined methods of hydrolysis. Chosen methods were physical, such as microwaves, increased temperature or ultrasound, and chemical acidic and alkaline. Combinations of both types were also examined. The last optimized method was enzymatic hydrolysis. First set of experiments was conducted using enzymes Novozymes® NS50013 and NS50010. Production of cellulase and pectinase enzymes by A. niger during solid-state fermentation that lasted 10 days was also studied. The yields of reducing sugars of all the experiments were calculated using the Somogyi-Nelson method. Enzymatic hydrolysis was proven to be the most effective using the combination of both of the enzymes for a period of 96 hours at pH = 4.5 and temperature 45 °C. Yield of the reducing sugars under these conditions reached 27,4241 ± 0,0007 gl-1.
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