Priprava in karakterizacija nanoteles 4T5 in 3T1

Zaveršek, Nika (avtor), Gunčar, Gregor (mentor)

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.

• Jezik: Slovenski jezik
• Ključne besede: nanotelo, enodomensko protitelo, koordinacija kovinskih ionov, dimerizacija, inkluzijska telesca
• Vrsta gradiva: Diplomsko delo/naloga
• Tipologija: 2.11 - Diplomsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2019
• PID: 20.500.12556/RUL-109943
• COBISS.SI-ID: 1538424003
• Datum objave v RUL: 10.09.2019

Sekundarni jezik

• Jezik: Angleški jezik

Izvleček

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.

• Ključne besede: nanobody, single-domain antibody, metal ion coordination, dimerisation, inclusion bodies