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Heterologous expression and purification of rat cytochrome P450 1A1
Vlková, Michaela ; Černá, Věra (advisor) ; Ingr, Marek (referee)
Cytochrome P450 1A1 belongs to "superfamily" of cytochromes P450 (CYPs). It is an extrahepatic enzyme that takes a part in detoxification metabolism of many xenobiotics but unfortunately also it is one of the most important representatives of CYPs involved in the activation of procarcinogens. The main aim of this bachelor thesis was to prepare and purify cytochrome P450 1A1 in sufficient quantity and purity by heterologous expression. Two different vectors, which were constructed by inserting the gene for rat cytochrome P450 1A1 in efficient expression plasmid pCW, were prepared during this bachelor thesis. Functionality of prepared vectors was verified by measuring the expression of CYP1A1 and CO difference spectra. It was found that only one of the vectors mentioned above provides native protein. This vector was transformed into E. coli cells of strain DH5α, DH5α with the inserted plasmid pHg1 and DH5α with inserted plasmid pGRO7. Various conditions of CYP1A1 production were tested: growth media (LB or TB medium), the time of production (24 and 48h), the concentration of IPTG (0.5mM and 1mM) and the presence of ALA. The highest expression level was achieved in E. coli DH5α cells cultivated in a modified TB medium after 48 hours of production and at 30řC (induction at OD600 0.6 to 0.8 by 0.5mM...
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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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Synthesis of 5'-UTR of the PSM-E variant of GCPII gene as a standard for RT-PCR
Růžičková, Barbora ; Ingr, Marek (advisor) ; Martínek, Václav (referee)
PCR method has recently become a key element for the syntesis of genes. In this work two-step DNA synthesis based on PCR was used in order to obtain 5'-UTR of the PSM-E splicing variant of glutamate carboxypeptidase II (GCPII) which should serve as a standard for qPCR in real time. Another aim of this work was to clone this sequence into the plasmid PCRII-TOPO PSM-E for the purpose of protein expression in insect cells in the baculovirus expression system. In the first PCR the middle section of the 5'-UTR sequence was synthesized. This product was subsequently amplified in the second PCR using the first and last oligonucleotide. The product of the second PCR was ligated into the plasmid pUC19 and E. coli was transformed by tis construct. Sequence correctness was confirmed by sequencing analysis. Subcloning of the insert from pUC19 to pCRII-TOPO PSM-E was carried out and the sequence was checked by sequencing analysis again. The obtained construct was used as a standard in qPCR in real time in order to determine the concentration of the samples of mRNA from the prostate tissues of oncologic patients
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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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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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Synthesis of the Var14 variant of 5'-UTR of GCPII gene as a standard for RT-PCR
Petrovová, Gabriela ; Ingr, Marek (advisor) ; Černá, Věra (referee)
The aim of this work was to prepare the synthetic 5'UTR sequence of splicing variant Var 14 of glutamate carboxypeptidase II (GCPII). A method of two-step PCA was used to this purpose. The sequence was divided into 8 overlapping oligonucleotides that were combined into a single dsDNA by two consecutive PCR. Product of the synthesis was cloned into the auxilliary cloning vector pUC19. After the sequencing analysis detected mutations were corrected. The product was subcloned into the target vector pcDNA4 His Var 14 which already contained the sequence GCPII gene. This construct was then used for the construction of the calibration curve, which will serve as a standard for RT-PCR for quantitative detection of this variant of GCP II in patients with prostate cancer. Construct will be further used as an expression vector to produce of the variants Var 14 GCPII in eucaryotic baculovirus expression system. Keywords: 5'UTR, two-step PCA, pUC19, RT-PCR, GCPII
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