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Preparation of recombinant human cytochrome b5
Hálková, Tereza ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a small heme protein. There are three isoforms present in human organism, one is located in the membrane of endoplasmic reticulum (microsomal cyt b5), second in the outer mitochondrial membrane and the third (soluble form) was found in cytoplasm of matured erythrocytes. The main role of cytochrome b5 is to transport single electron in various reactions including cytochrome P450-dependent reactions. First aim of the thesis was to prepare and to isolate the soluble form of rabbit microsomal cytochrome b5, using heterologous expression in Escherichia coli strain BL-21 Gold. The plasmid pET22b containing synthetic gene for rabbit microsomal cytochrome b5, lacking the sequence encoding the membrane associated C-terminal domain, was used as an expression vector. The second aim was to synthesize the expression vectors carrying genes for human microsomal and erythrocytic cytochromes b5. These genes were prepared by gene synthesis, ligated to cloning vector pUC19, amplified in E. coli DH5α competent cells and their sequences were verified by DNA sequencing. Consequently the pET22b expression vectors containing genes for human microsomal and erythrocytic cytochrome b5 were constructed and finally their suitability for heterologous expression was evaluated. Keywords: Heterologous expression,...
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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Effect of gene optimization on recombinant expression of human cytochrome P450 3A4
Svobodová, Barbora ; Martínek, Václav (advisor) ; Bořek Dohalská, Lucie (referee)
Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis
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(Construction of deletion mutants of human cytochrome b5 using gene synthesis)
Kotlánová, Iveta ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochrome b5 is a small amphipathic protein. The human form is anchored to the outer membrane of the endoplasmic reticulum and mitochondria, a free form is located in red blood cells. It consists of two domains: a large hydrophilic domain binds heme, a small hydrophobic domain anchors cytochrome b5 to the microsomal membrane. Both domains are connected by linker chain of about 15 amino acids, which gives a flexibility to the protein. Its length plays an important role in transferring electrons to cytochrome P450. If the linker domain is too short, cytochrome b5 is not able to tranfer electrons to cytochrome P450 and not participates in the reactions of MFO system. Other functions are preserved. The aim of this study was to design and build 4 deletion mutants of cytochrome b5 using gene synthesis. The linker domain contains long and short deletions, which are expected to have distortion interaction with cytochrome P450. Part of this thesis was the expression of heterologous proteins by cells of Escherichia coli strain XL10-Gold and DH5α. As expression vectors for the transformation were used plasmids pET- 30a(+) and pET-22b. DNA from cells was isolated and the accuracy of the genetic code was verified using the sequencing. Keywords: cytochrome b5, heterologous expression, gene synthesis (In Czech)
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(Construction of deletion mutants of human cytochrome b5 using gene synthesis)
Kotlánová, Iveta ; Martínek, Václav (advisor) ; Moserová, Michaela (referee)
Cytochrome b5 is a small amphipathic protein. The human form is anchored to the outer membrane of the endoplasmic reticulum and mitochondria, a free form is located in red blood cells. It consists of two domains: a large hydrophilic domain binds heme, a small hydrophobic domain anchors cytochrome b5 to the microsomal membrane. Both domains are connected by linker chain of about 15 amino acids, which gives a flexibility to the protein. Its length plays an important role in transferring electrons to cytochrome P450. If the linker domain is too short, cytochrome b5 is not able to tranfer electrons to cytochrome P450 and not participates in the reactions of MFO system. Other functions are preserved. The aim of this study was to design and build 4 deletion mutants of cytochrome b5 using gene synthesis. The linker domain contains long and short deletions, which are expected to have distortion interaction with cytochrome P450. Part of this thesis was the expression of heterologous proteins by cells of Escherichia coli strain XL10-Gold and DH5α. As expression vectors for the transformation were used plasmids pET- 30a(+) and pET-22b. DNA from cells was isolated and the accuracy of the genetic code was verified using the sequencing. Keywords: cytochrome b5, heterologous expression, gene synthesis (In Czech)
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Preparation of recombinant cytochrome P450 1A1
Dvořák, Martin ; Svášková, Dagmar (advisor) ; Ingr, Marek (referee)
5 Abstract Cytochrome P450 1A1 (CYP1A1) is one of the major isoforms of the cytochrome P450 superfamily. It is primarily an extrahepatic enzyme which is responsible for oxidation of many polycyclic aromatic hydrocarbons and other xenobiotics. Besides of the role in detoxification metabolism CYP1A1 is the one most important isoform involved in activation of procarcinogens. The main aim of this project was preparation of two modifications of the rat CYP1A1 gene with codon optimization for expression in E. coli by gene synthesis. One was wild type (wt1A1) and the other was with modified N-terminal anchor (mod1A1) - for both modifications with or without His Tag at the C-end of CYP1A1. Furthermore, an aim was to compare their level of expression in different strains of E. coli and try to purify and assess enzymatic activity of the gene's products. From pre-prepared oligonucleotides 2 "syntons" (parts of gene) were synthetized and separately inserted into pUC19. After verified sequence of the "syntons" they were cleaved from pUC19 and inserted together into pET-22b. These vectors were prepared for transformation of 3 strains of E. coli (BL-21 (DE3) GOLD, RIL a RIPL). For production of proteins many conditions were tested: temperature (18, 22, 24, 27 a 37 řC), time of production (untill 48 hours), concentration...
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Effect of gene optimization on recombinant expression of human cytochrome P450 3A4
Svobodová, Barbora ; Martínek, Václav (advisor) ; Bořek Dohalská, Lucie (referee)
Cytochrome P450 3A4 is integral membrane protein residing in endoplasmic reticular membrane. In human the highest concentration cytochrome P450 3A4 is expressed in liver, where it plays a major role in metabolism of many drugs and xenobiotics. The main aim of the thesis was to evaluate the effect of gene optimization on heterologous expression of human cytochrome P450 3A4. At first expression constructs based on vectors pET22b were prepared. Then the efficiency of heterologous expression of optimized vs. natural gene sequence encoding truncated form of the human cytochrome P450 3A4 compared. The results show that the gen sequence optimized for E. coli strains K12 was expressed in significantly higher efficiency than the original human gene based on cDNA sequence. Another aim was to evaluate the suitability of pET22b based expression vectors for recombinant production of native (complete) form of human cytochrome P450 3A4. The amount of native form of the protein found in bacterial membrane was however substantially lower then that of the truncated form. Keywords: cytochrome P450 3A4, heterologous expression, pET22b, gene synthesis
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Preparation of expression system of gamma-lactamase and expression testing
Magyerková, Monika ; Ingr, Marek (advisor) ; Šácha, Pavel (referee)
γ-lactamase is an enzyme clearing five-membered lactam cycles. Polyvinylpyrrolidone (PVP) is one of its potential substrates. Degradation of PVP by γ-lactamase is being studied due to its eventual use in waste-water purifying plants. The aim of the work was to prepare a synthetic gene from the bacterium Comamonas acidovorans and to clone it into the expression vector pET22b. PCA method was used for the synthesis of the γ-lactamase gene. 1725 bp long sequence of the γ-lactamase gene was split into two parts (synthons) which were synthesized individually. After the synthesis restriction cleavage and ligation to the vector pUC19 were performed. Competent cells E. coli, strain DH5α, were transformed by the obtained construct. After the sequence confirmation both synthons were cleaved by restriction endonucleases and connected by single-step ligation to the plasmid pET22b. Expression bacterial cells E. coli, strain BL21(DE3)RIL, were transformed by the recombinant plasmid containing the connected synthons and expression of the recombinant γ-lactamase was tested. Sequence of the clone producing a protein of the expected length was confirmed by sequencing analysis. The prepared plasmid will be used for the expression of recombinant γ- lactamase. (In English)
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Preparation of recombinant human cytochrome b5
Hálková, Tereza ; Martínek, Václav (advisor) ; Hudeček, Jiří (referee)
Cytochrome b5 is a small heme protein. There are three isoforms present in human organism, one is located in the membrane of endoplasmic reticulum (microsomal cyt b5), second in the outer mitochondrial membrane and the third (soluble form) was found in cytoplasm of matured erythrocytes. The main role of cytochrome b5 is to transport single electron in various reactions including cytochrome P450-dependent reactions. First aim of the thesis was to prepare and to isolate the soluble form of rabbit microsomal cytochrome b5, using heterologous expression in Escherichia coli strain BL-21 Gold. The plasmid pET22b containing synthetic gene for rabbit microsomal cytochrome b5, lacking the sequence encoding the membrane associated C-terminal domain, was used as an expression vector. The second aim was to synthesize the expression vectors carrying genes for human microsomal and erythrocytic cytochromes b5. These genes were prepared by gene synthesis, ligated to cloning vector pUC19, amplified in E. coli DH5α competent cells and their sequences were verified by DNA sequencing. Consequently the pET22b expression vectors containing genes for human microsomal and erythrocytic cytochrome b5 were constructed and finally their suitability for heterologous expression was evaluated. Keywords: Heterologous expression,...
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