Purifikácia membránovo viazaného enzýmu DHRS7B ; Purification of membrane bound enzyme DHRS7B ; Purifikace membránově vázaného enzymu DHRS7B

Charles University Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Valéria Mariničová Supervisor: RNDr. Eva Novotná , Ph.D. Title of diploma thesis: The purification of the membrane-bound enzyme DHRS7B The membrane-bound DHRS7B enzyme, from the SDR (short-chain dehydrogenase/reductase) superfamily, is one of poorly characterized protein of the human organism. Despite the lack of information about its properties, it was observed in mice, where it is named to as PexRap, localized in peroxisomes. It is a peroxisomal reducing protein that uses NADPH as a cofactor. Its participates in biosynthesis of peroxisomal ether lipids during adipogenesis in mice. The role of human DHRS7B in cells or evantually in diseases has been only speculated and therefore it is in the focus of research. The aim of this work was to obtain the pure recombinant human DHRS7B enzyme prepared in Sf9 cells. It is a membrane protein, so it was necessary to find a suitable detergent that is able to remove the DHRS7B enzyme from the phospholipid membrane while preserving its activity. The most suitable detergent was DDM 1:1 (w/w) protein:detergent. Further step was its purification by affinity chromatography, the pure DHRS7B protein with a ketotifen reducing activity of 62 nmol/mg/min was obtained. ; Univerzita Karlova Farmaceutická fakulta v Hradci Králové Katedra biochemických věd Kandidát: Valéria Mariničová Vedúca diplomovej práce: RNDr. Eva Novotná, Ph.D. Názov diplomovej práce: Purifikácia membránovo viazaného enzýmu DHRS7B Membránovo viazaný enzým DHRS7B, pochádzajúci z nadrodiny SDR (dehydrogenázy/reduktáz s krátkym reťazcom), patrí medzi málo preskúmané proteíny ľudského organizmu. Aj napriek nedostatku informácii o jeho vlastnostiach bol pozorovaný u myší, kde sa označuje ako PexRap s lokalizáciou v peroxizomoch a endoplazmatickom retikule (ER). Ide teda o peroxizomálny redukujúci proteín, ktorý ako kofaktor využíva NADPH. V myšacom organizme sa prejavila jeho aktivita na biosyntéze peroxizomálnych ...