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Priprava in karakterizacija nanoteles 4T5 in 3T1
Zaveršek, Nika (Author), Gunčar, Gregor (Mentor) More about this mentor... This link opens in a new window

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Abstract
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.

Language:Slovenian
Keywords:nanotelo, enodomensko protitelo, koordinacija kovinskih ionov, dimerizacija, inkluzijska telesca
Work type:Bachelor thesis/paper (mb11)
Tipology:2.11 - Undergraduate Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2019
COBISS.SI-ID:1538424003 Link is opened in a new window
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Downloads:16
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Secondary language

Language:English
Abstract:
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.

Keywords:nanobody, single-domain antibody, metal ion coordination, dimerisation, inclusion bodies

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