Načrt in karakterizacija inducibilne dimerizacije svežnja štirih vijačnic v sesalski celični liniji HEK293T

magistrsko delo

Benjamin Malovrh (Author), Roman Jerala (Mentor), Gregor Gunčar (Thesis defence commission member), Vera Župunski (Thesis defence commission member), Janez Plavec (Co-mentor)

Abstract

Sintezna biologija je veda, ki se ukvarja z načrtovanjem novih biomolekularnih komponent, signalnih poti in kaskad, ter uporabo le-teh za uravnavanje delovanja celic, reprogramiranje organizacije tkiv in organizmov. Primer takšnega modula je tudi de novo načrtovan sintetični protein, ki tvori motiv svežnja štirih vijačnic (angl. four-helical bundle). Modul sestavljajo štiri α-vijačnice obvite med seboj. De novo načrtovani svežnji štirih vijačnic imajo lahko aplikativno vrednost tudi v sesalskih celičnih linijah kot nove dimerizacijske, trimerizacijske ali tetramerizacijske domene. Z ustreznim načrtovanjem sistema smo pokazali, da je mogoče regulirati inducibilno 1:3 dimerizacijo α-vijačnic v sveženj štirih vijačnic, preko cepitve z eno izmed ortogonalnih virusnih proteaz. Katalitično aktivna proteaza cepi vstavljeno cepitveno mesto med dimerizacijskimi moduli svežnja štirih vijačnic in s tem sproži dimerizacijo. Virusne proteaze smo uporabili tudi v razcepljeni neaktivni obliki, aktivnost katerih se ob ustrezni prostorski razporeditvi povrne. Prostorsko približevanje cepljenih domen smo izvedli preko sistema kemijsko uravnane dimerizacije dveh različnih domen, ki imata preko peptidnega povezovalca vezane domene razcepljenih proteaz. Ob dodatku ustreznega kemijskega induktorja, je prišlo do dimerizacije domen receptorja za induktor, prostorskega približevanja cepljenih proteaznih domen in s tem povrnitve katalitične aktivnosti. Časovna odzivnost pripravljenega sistema je hitrejša od transkripcijske regulacije in omogoča štirikratno povišanje signala v časovnem razponu 2 h. Uspešnost dimerizacije svežnja štirih vijačnic smo preverjali s povrnitvijo luciferazne aktivnosti. Luciferazni reporter je mogoče nadomestiti z drugimi celičnimi komponentami in tako preko svežnja štirih vijačnic uravnavati ali zaznavati številne celične procese.

Keywords

proteini;proteinski moduli;de novo;sveženj štirih vijačnic;inducibilna dimerizacija;časovna odzivnost;proteaze;magistrska dela;

Data

Language:	Slovenian
Year of publishing:	2021
Typology:	2.09 - Master's Thesis
Organization:	UL FKKT - Faculty of Chemistry and Chemical Technology
Publisher:	[B. Malovrh]
UDC:	577.112(043.2)
COBISS:	90909187
Views:	196
Downloads:	5
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	Design and characterization of inducible dimerization of four-helix bundle in mammalian cell line HEK293T
Secondary abstract:	Synthetic biology is a field of science that deals with design of new biomolecular components, signalling pathways, cascades, and their use to regulate cell function, reprogramming tissue, and organism organizations. An example of a module is de novo designed synthetic protein, which forms the motif of a four helical bundle. The module is represented by four α-helices wrapped together. De novo designed four helical bundles also have applicative use in mammalian cell lines as dimerization, trimerization or tetramerization modules. With proper system design, we have shown that it is possible to regulate the inducible 1: 3 dimerization of α-helices in four helical bundle that can be regulated via orthogonal viral proteases. The catalytically active protease cleaves the inserted cleavage site between the dimerization modules of the four helical bundle and thus induces dimerization. Viral proteases were also used in a cleaved inactive form, the activity of which is restored with appropriate proximity manipulation. Spatial approximation was performed via a system of exogenously regulated dimerization of heterodimerization domains having linker-linked cleaved protease units. With the addition of an appropriate chemical inducer, the dimerization domains bind. This results in the spatial approximation of the protease units, and thus the restoration of its catalytic activity. Time response of the prepared system is faster than the transcriptional regulation. And it allows a fourfold increase in signal over a time span of 2 h. The inducible dimerization of the four helical bundle was designed and characterized on the luciferase reporter system. The luciferase reporter can be replaced with other cellular components and thus regulate or detect several cellular processes via four helical bundle.
Secondary keywords:	de novo;four helical bundle;inducible dimerization;time response;
Type (COBISS):	Master's thesis/paper
Study programme:	1000377
Embargo end date (OpenAIRE):	2022-12-17
Thesis comment:	Univ. v Ljubljani, Fak. za kemijo in kemijsko tehnologijo, smer Biokemija
Pages:	52 str.
ID:	14562754