Glycoprotein erythropoietin is a major humoral factor in the body, which, by promoting erythropoiesis, increases the oxygen carrying capacity of blood. Such an increase has a positive effect on physical endurance, which, despite being banned by the World Anti-Doping Organization, has led some athletes to resort to the use of erythropoietin or substances that mimic its physiological action. Proving the presence of such, especially low-molecular weight substances, is, for the time being, quite difficult and unreliable, and there is a tendency to develop a universal testing platform. In the development of such a test platform, one of the components used was the recombinant extracellular region of the erythropoietin receptor (EpoR), which was obtained by transient expression in mammalian cells (the HEK 293T cell line). This kind of expression is demanding, time consuming and expensive, so we wanted to find an easier, faster and cheaper way to produce EpoR. The purpose of the master's thesis was to experimentally obtain EpoR using E. coli as an alternative expression system to mammalian HEK 293T cells. A major obstacle to bacterial expression systems is that they do not provide the oxidizing environment in the cytoplasm that EpoR requires for proper folding and formation of disulfide bridges. We tried to overcome this limitation by attaching a gene sequence encoding a signal peptide to the 5'-end of the sequence coding for EpoR, which, after expression, directs the polypeptide chain into the periplasmic space, which has an oxidative environment. After infection of E. coli with bacteriophage M13 particles harbouring phagmid vector pIT2/EpoR, test expression of EpoR under different induction conditions at two different temperatures was first carried out to determine the optimal conditions for EpoR expression. Then, bulk expression was performed and the product was purified by immobilized metal affinity chromatography and size exclusion chromatography. Finally, the product yield was evaluated colorimetrically using the Bradford assay, spectrophotometrically at 280 nm and densitometrically, and its activity was evaluated using a modified version of the enzyme-linked immunosorbent assay (ELISA) and compared with the activity of EpoR obtained by expression in mammalian HEK 293T cells. Both the concentration (~150 μg / 1 L of culture) and the activity of expressed EpoR (~0.4% compared to almost 100% activity of EpoR expressed in HEK 293T) were very low.
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