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Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Tereza Lundová Supervisor: Prof. Ing. Vladimír Wsól, Ph.D. Title of diploma thesis: Cloning, expression and purification of human AKR1B1 Aldose reductase (EC 1.1.1.21) AKR1B1 is one of the 13 human enzymes of the AKR superfamily. All human AKRs are cytosolic and NADP(H) dependent. AKR1B1 plays an important role in metabolism of endogenous and exogenous substances. The main endogenous substrate is glucose. Its reduction to sorbitol is consistently linked to secondary diabetic complications. From xenobiotics metabolized by AKR1B1, daunorubicin, an anticancer drug from the group of anthracyclines, is reduced to daunorubicinol. This metabolite is less active than parent drug and is the cause of anthracycline related cardiotoxicity. At present, many projects are focused on AKR1B1 as a target enzyme and specific inhibitors of AKR1B1 are looking for. The recombinant protein of AKR1B1 was prepared in E. coli together with the pET expression system. First, cDNA for AKR1B1 in pOTB7 was isolated from E. coli. The coding sequence of AKR1B1 was amplified by a PCR. PCR was performed with Phusion Hot Start II polymerase and pair of forward and reverse primers, which contained NdeI and XhoI restriction... |
