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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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Heterologous expression of human NADPH:cytochrome P450 reductase
Mazurová, Martina ; Martínek, Václav (advisor) ; Ingr, Marek (referee)
Study of carcinogenesis is associated with study of xenobiotics metabolism, which is topic studied in our laboratory. Mixed-function oxygenase system (MFO system) is significantly contributing to the metabolism of xenobiotics. Pure recombinant proteins participating in MFO system are frequently utilized in in vitro metabolic experiments. The heterologous expression method is often used to obtain the pure recombinant enzymes. Heterologous expression was employed to prepare human NADPH:cytochrome P450 oxidoreductase. This membrane enzyme reduces cytochrome P450 and enables its catalytic activity. Vectors with synthetic gene for human NADPH:cytochrome P450 oxidoreductase based on pUC19 and pET22b plasmids were prepared and verified. Recombinant protein was produced in E. coli BL21-Gold and E. coli BL21-CodonPlus-RIL cells. Both cell strains produced high levels of the protein; however the major part of the protein was present predominantly in inclusion bodies. Expression conditions were therefore optimized to obtain higher yields of native protein bound in bacterial membrane fraction. [In Czech]
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Structure and interaction of human 14-3-3 regulatory protein: photoaffinity labelling in vitro and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Kavan, Daniel (referee)
This work is focused on the interactome study of 14-3-3ζ protein, a regulatory protein found in all eucaryotic cells. An important 14-3-3 protein feature is the ability to bind a number of structurally and functionally distinct protein ligands. This link is usually implemented through phosphorylated serine and threonine motifs. The first aim of this work is the preparation of sufficient amount of recombinant 14-3-3ζ protein with incorporated photoactivatable analogue of methionine (foto-Met, L-2-amino-5,5- azihexan acid). The four different conditions of recombinant expression in auxotrophic E. coli B834 (DE3) strain were tested to obtain a protein with a maximal rate of photoactivatable methionine analogue incorporation into the sequence 14-3-3 protein. The second aim is to study the methionine 121, 160 and 218 participation in the 14-3-3ζ protein binding groove and finding of potential covalent bond with the phosphorylated peptide 251-266 of Raf-1 kinase (phosphorylation on Ser259). The photo-initiated cross-linking method was used (photolysis), to form a reactive biradical of methionine analogue capable to attack any amino acid residues in close vicinity (till 5Å). Finally, the products of photo-initiated cross-linking were analyzed by cross-linking reactions using MALDI-TOF MS, LC-MS and...
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Structure and interaction of human 14-3-3 regulatory protein using in vitro photoaffinity labelling in combination of protein nano-probes and mass spectrometry
Mazurová, Martina ; Šulc, Miroslav (advisor) ; Dračínská, Helena (referee)
This thesis is focused on the study of the structure and mechanism of human 14- 3-3 protein, which is one of the important regulatory proteins present in all eukaryotic cells. Nowadays it is known seven isoforms of this protein in mammals. Although their crystal structure shows a high similarity, their mutual comparison reveals some changes. The aim of this work is to prepare experimental tools for verification whether the differences in the crystal structure of the ζ isoform are present in solution and how the structure-functional mechanism of this isoform is affected. The otimization of 14-3- 3zeta recombinant protein expression with incorporated a photo-labile analog of leucine in the protein sequence was performed using limiting medium with prokaryotic expression system of E. coli BL-21 DE3 Gold or system of auxotrophic E. coli K-12 with non-functional leucine biosynthesis.
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Heterologous expression of human NADPH:cytochrome P450 reductase
Mazurová, Martina ; Martínek, Václav (advisor) ; Ingr, Marek (referee)
Study of carcinogenesis is associated with study of xenobiotics metabolism, which is topic studied in our laboratory. Mixed-function oxygenase system (MFO system) is significantly contributing to the metabolism of xenobiotics. Pure recombinant proteins participating in MFO system are frequently utilized in in vitro metabolic experiments. The heterologous expression method is often used to obtain the pure recombinant enzymes. Heterologous expression was employed to prepare human NADPH:cytochrome P450 oxidoreductase. This membrane enzyme reduces cytochrome P450 and enables its catalytic activity. Vectors with synthetic gene for human NADPH:cytochrome P450 oxidoreductase based on pUC19 and pET22b plasmids were prepared and verified. Recombinant protein was produced in E. coli BL21-Gold and E. coli BL21-CodonPlus-RIL cells. Both cell strains produced high levels of the protein; however the major part of the protein was present predominantly in inclusion bodies. Expression conditions were therefore optimized to obtain higher yields of native protein bound in bacterial membrane fraction. [In Czech]
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