GPx is a known antioxidant enzyme abundant in human body cells which drives the H2O2 scavenge into water.
<liGlutathione (GSH) is a peptide consisting of three amino acid residues, glutamate, cysteine and glycine, respectively. It is a natural cellular component for preventing damage to organelles caused by reactive oxygen species, free radicals or peroxides.
In cell, GSH appears in different components or biochemical structures. GSH is a cofactor for GPx enzyme that the thiol group of cysteine in GSH is able to donate a reducing equivalent (H++ e−) to other unstable molecules, such as reactive oxygen species. In donating an electron, GSH itself becomes reactive, but readily reacts with another reactive GSH to form glutathione disulfide. (GSSG)
GSH can be regenerated from GSSG by the enzyme glutathione reductase (GSR) which uses NADPH cofactor. The scheme of reduction cascade can be seen in figure X:
In literature, GPx-1 can be overexpressed in transfected cells and produced synthetically. This recombinant enzyme is shown to work against hydrogen peroxide, lipid peroxide and drugs interfering with redox cycle
GENE SYNTHESIS FROM cDNA OF 64A CELL LİNE
GPx inserts were acquired from the ordered genes and cDNA. The results showed the expected range of 500-750 bp.
CLONING CONTROL-1
Colony PCR was conducted using the specified primers. As it can be seen on the figure, the results were not accurately fitting the base pair range mentioned. The experiment was repeated several times, and after unsuccessful attempts, the parts were ordered for artificial synthesis.
CLONING CONTROL-2
The ligated and transformated samples were put into colony PCR. As it can be seen on the figure, the right inserts were located in colonies 7,8 and 10.
WESTERN BLOTTİNG
Sadly, the results achieved did not fit the ideal Blotting results.
WESTERN BLOTTİNG
Selenium was necessary for the production of the selenocysteine amino acid present in GPx but was not present in the medium of the culture. It was expected to have observed a result such as the figure on the left through adding Na2SeO3 to the cell line. Expression was seen; there was a directly proportional increase in GPx production in respect to selenoite increase(100 uM-200 uM-500 uM-1000 uM).
The bands acquired through the Western Blotting were fitting the expected results. The cell lines, that now had their transfections verified, were purfied for the preparation of GPx assays.
The amounts of protein acquired were as the charts above and the these data were taken into consideration in the preparation of the assays.
A functional assay of GPx was done through NADPH to measure GPx activity. The same amount of genes were transferred to all samples but varying amount of Na2SeO3 was prepared in the samples, with 2 samples containing different Na2SeO3 concentrations and one control group. The GPx gene was not transfected to the control group. From these 3 groups, the assay of the control group gave the lowest yield as expected; the sample of 500 nM Na2SeO3 produced the mid-result. The sample of 1000 nM Na2SeO3 yielded the highest activity results in the assay. These results prove that the GPx production is directly proportional to the amount of added Na2Se2O3 and that the GPx enzyme was produced functionally.
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ATOMS TURKİYE 2014
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