diplomsko delo
Nika Zaveršek (Author), Gregor Gunčar (Mentor)

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.

Keywords

nanotelesa;enodomensko protitelo;koordinacija kovinskih ionov;dimerizacija;inkluzijska telesca;diplomska dela;

Data

Language: Slovenian
Year of publishing:
Typology: 2.11 - Undergraduate Thesis
Organization: UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher: [N. Zaveršek]
UDC: 577.27(043.2)
COBISS: 1538424003 Link will open in a new window
Views: 828
Downloads: 299
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Other data

Secondary language: English
Secondary 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.
Secondary keywords: nanobody;single-domain antibody;metal ion coordination;dimerisation;inclusion bodies;
Type (COBISS): Bachelor thesis/paper
Study programme: 1000371
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo
Pages: 40 str.
ID: 11221113