20.500.12556/RUL-109943 Priprava in karakterizacija nanoteles 4T5 in 3T1 Nanotelesa so variabilne domene HCAb (angl. heavy chain antibody), ki jih najdemo v družini Chamelidae. So majhna, stabilna, v obliki samostojne domene, imajo veliko afiniteto do antigena in jih je enostavno izražati v bakterijskih sistemih. Zaradi svojih dobrih lastnosti na veliko področjih prekašajo in izpodrivajo navadna tetramerna protitelesa. V diplomskem delu smo raziskovali dva mutanta nanotelesa M33 z afiniteto do MLKL. Mutanta, nanotelesi 4T5 in 3T1, sta bili pripravljeni tako, da bi lahko tvorili homodimer s pomočjo kobaltovih ionov. Avtoindukcijsko smo ju izrazili v bakterijskem sistemu BL21[DE3]. Izolirali smo ju iz inkluzijskih telesc in ju renaturirali ter očistili s pomočjo histidinske oznake s kolono IMAC na napravi FPLC. Oznako His smo odcepili s proteazo TEV. Nanotelesi 4T5 in 3T1 smo nanesli na kolono za kromatografijo z ločevanjem po velikosti, nato pa smo na kolono za kromatografijo z ločevanjem po velikosti nanesli še nanotelo 4T5 v prisotnosti kobaltovih ionov, da bi ugotovili, če pride do dimerizacije. Tvorba homodimera pod uporabljenimi pogoji ni potekla. Nanobodies are HCAb derived single-domain antibodies found in Chamelidae serum. Their small size, stability, high antigen affinity and ability of expression in bacterial systems are useful assets in research areas, where affinity is a useful property. We used two M33 nanobody mutants, which have MLKL affinity. Mutant nanobodies 4T5 and 3T1 were designed to form a homodimer in the presence of cobalt ions. Expression by autoinduction was performed in BL21[DE3]. Nanobodies were isolated from inclusion bodies and later refolded by sulfonation and renaturation. Purification was done via His-tag and IMAC on FPLC. We removed His-tag by TEV protease cleavage. Size exclusion liquid chromatography was used to test the dimerization of 4T5 nanobody, which did not occur under the conditions that were used. nanotelo enodomensko protitelo koordinacija kovinskih ionov dimerizacija inkluzijska telesca nanobody single-domain antibody metal ion coordination dimerisation inclusion bodies true false false Slovenski jezik Angleški jezik Diplomsko delo/naloga 2019-09-10 14:45:08 2019-09-10 14:45:10 2022-08-22 03:40:39 0000-00-00 00:00:00 2019 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 6423 1538424003 Zaversek_Nika_-_Priprava_in_karakterizacija_nanoteles_4T5,_3T1.pdf Zaversek_Nika_-_Priprava_in_karakterizacija_nanoteles_4T5,_3T1.pdf 1 DCD6C1B451ECD9824230533A8CDCAC73 5c343b081d8059d4266ead3b6b8a0b504e3f06c39eae1a93aab5182fa6c3ed58 fef88916-a1b6-11eb-a523-00155dcfd717 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=121549 Fakulteta za kemijo in kemijsko tehnologijo 0 0 0