20.500.12556/RUL-114407 Izražanje rekombinantne zunajcelične regije človeškega eritropoetinskega receptorja v periplazmi bakterije Escherichia coli Expression of recombinant human erythropoietin receptor ectodomain in the periplasm of Escherichia coli Glikoprotein eritropoetin je glavni humoralni dejavnik v telesu, ki s spodbujanjem eritropoeze povečuje oksiformno kapaciteto krvi. Takšno povečanje pozitivno vpliva na fizično vzdržljivost, zaradi česar se, kljub prepovedi s strani Svetovne protidopinške organizacije, nekateri športniki zatekajo k uporabi eritropoetina oz. substanc, ki oponašajo njegovo fiziološko delovanje. Dokazovanje prisotnosti takšnih – zlasti nizkomolekularnih – spojin je zaenkrat precej težavno in nezanesljivo, zaradi česar obstaja težnja po razvoju univerzalne testne platforme. Pri razvoju takšne testne platforme je bila ena izmed uporabljenih komponent tudi rekombinantna zunajcelična regija eritropoetinskega receptorja (EpoR), ki so jo pridobili s prehodnim izražanjem v sesalski celični liniji HEK 293T. Takšno izražanje je zahtevno, dolgotrajno in drago, zato smo želeli najti enostavnejši, hitrejši in cenejši način pridobivanja EpoR. Namen magistrske naloge je bilo poskusno pridobivanje EpoR z uporabo E. coli kot alternativnega ekspresijskega sistema sesalskim celicam HEK 293T. Glavna ovira bakterijskih ekspresijskih sistemov je, da v citoplazmi ne nudijo oksidacijskega okolja, ki ga EpoR potrebuje za pravilno zvijanje in tvorbo disulfidnih mostičkov. To omejitev smo poskusili odpraviti tako, da smo genskemu zapisu za EpoR na 5'-konec pripojili genski zapis za signalno zaporedje, ki po izražanju usmerja polipeptidno verigo v periplazemski prostor z oksidativnim okoljem. Po okužbi E. coli z bakteriofagnimi delci M13, ki so vsebovali fagmidni vektor pIT2/EpoR, smo, da bi določili optimalne pogoje za izražanje EpoR, najprej izvedli testno izražanje EpoR v različnih pogojih indukcije pri dveh različnih temperaturah. Nato smo izvedli izražanje v večji količini in produkt očistili s kovinsko-kelatno afinitetno kromatografijo in velikostno-izključitveno kromatografijo. Na koncu smo količino produkta ocenili kolorimetrično po Bradfordu, z merjenjem absorbance pri 280 nm in denzitometrično ter ocenili njegovo aktivnost s prilagojeno različico testa ELISA in jo primerjali z aktivnostjo EpoR, pridobljenega z izražanjem v sesalskih celicah HEK 293T. Tako koncentracija (~150 μg / 1 L kulture) kot aktivnost izraženega EpoR (~0,4 % v primerjavi s skoraj 100 % aktivnostjo EpoR, izraženega v HEK 293T) sta bili zelo nizki. 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. Escherichia coli periplazemsko izražanje eritropoetinski receptor Escherichia coli periplasmic expression erythropoietin receptor true false false Slovenski jezik Angleški jezik Magistrsko delo/naloga 2020-02-27 08:35:27 2020-02-27 08:35:35 2022-08-26 03:50:21 0000-00-00 00:00:00 2020 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 72791 918.pdf 918.pdf 1 78BEE0EEC7212D88ABC9A5166F6A8C7D d8aa6f1ae79b17f1b8ce81edb33e67eab3e6ef3ce8d13e1c2c06f7bdbdaa4f51 36a42e32-a1b8-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=126587 Fakulteta za farmacijo 0 0 0