20.500.12556/RUL-103373 Kvasni peroksin Pex11 in njegova predpostavljena funkcija transporta v membrani unilamelarnih veziklov Yeast peroxin pex11 and its proposed transporter function in membranes of unilamellar vesicles Peroksin-11 (Pex11) je membranski protein, ki se nahaja na peroksisomih kvasovke Saccharomyces cerevisiae. Nedavno je bilo predpostavljeno, da naj bi v perkosisomih tvoril pore za prehajanje topljencev manjših od 400 Da. Namen naloge je bil nadgraditi omenjeno raziskavo, pri čemer smo želeli (i) pridobiti rekombinantni Pex11 in preveriti njegovo porotvorno aktivnost na unilamelarnih veziklih napolnjenih s kalceinom ter (ii) preveriti specifičnost pore za prehajanje molekul NADH in koencima A na sistemu planarnih membran. Za pridobivanje Pex11 smo testirali dva različna seva in vektorja ter ovrednotili donos rekombinantnega Pex11 pri različnih pogojih gojenja. Optimiziran postopek gojenja smo prenesli na večjo skalo in uspešno izolirali rekombinatni Pex11. Z namenom doložitve lipidnega vezavnega partnerja smo izvedli sedimentacijski test in točkovni nanos lipidov. Porotvornost smo preverjali s kalceinskim testom na unilamelarnih veziklih, sestavljenih iz celokupnega lipidnega ekstrakta govejih eritrocitov ali fosfatidilholina. Čeprav lipidnega receptorja in specifičnosti pore nismo uspeli določiti, smo z vezikli iz celokupnega lipidnega ekstrakta govejih eritrocitov potrdili, da je Pex11 porotovrni protein. Peroxin 11 (Pex11) is a peroxisome membrane protein of the yeast Saccharomyces cerevisiae. It has recently been assumed that Pex11 forms pores that allow the passage of solutes smaller than 400 Da. The aim of the thesis was to upgrade the mentioned study, by (i) obtaining recombinant Pex11 and verifying its pore-forming activity on unilamellar vesicles loaded with calcein, and (ii) checking the specificity of the pore for the transport of NADH and coenzyme A using the system of planar membranes. To obtain Pex11, two different strains and vectors were tested and evaluated for the yield of recombinant Pex11 under different cultivation conditions. The optimized cultivation process was transferred to a larger scale and we successfully isolated recombinant Pex11. In order to determine the lipid binding partner, we performed a sedimentation test and a lipid dot blot assay. The pore-forming activity was tested fluorimetrically on calcein-loaded unilamellar lipid vesicles composed of a total lipid extract of bovine erythrocytes or phosphatidylcholine. Although we were not able to determine the lipid receptor and pore specificity, vesicles from the total lipid extract of bovine erythrocytes confirmed, that Pex11 was a pore-forming protein. biologija Saccharomyces cerevisiae kvasovke peroksisomi peroksini Pex11 biology Saccharomyces cerevisiae yeasts peroxisomes peroxins Pex11 true false false Slovenski jezik Neznan jezik Magistrsko delo/naloga 2018-09-16 07:47:22 2018-09-16 07:47:23 2022-08-16 03:41:42 0000-00-00 00:00:00 2018 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 156410 4925007 3809.pdf 3809.pdf 1 80BA901EBFAF44A1AC93DB702A823AD4 3751df4361f9bd32d994fb357a8194ef146d14be0a73c23ca791a2e602852960 c9ef3a41-a1b5-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=113792 Biotehniška fakulteta 0 0 0