Izolacija - rekombinantnega flagelina

diplomsko delo visokošolskega strokovnega študijskega programa I. stopnje

Robert Bremšak (Author), Uroš Potočnik (Mentor), Karolina Ivičak (Co-mentor)

Abstract

Tollu-podobni receptorji (ang. Toll-like receptors oz. TLRs) so transmembranski receptorji tipa I, sestavljeni iz ektodomene, transmembranske domene in znotrajcelične signalne domene TIR (ang. Toll/interleukin-1 receptor domain). TLR-ji prepoznavajo molekulske vzorce, značilne za mikroorganizme in endogene signale nevarnosti, ki sprožijo imunski odgovor, ki vključuje vnetje in produkcijo citokinov. Toll-u podobni receptor 5 oz. TLR5 prepoznava flagelin, ki je glavna sestavina bakterijskih bičkov in virulenčni dejavnik številnih po Gramu pozitivnih in po Gramu negativnih bakterij. Prepoznavanje flagelina je evolucijsko ohranjen mehanizem. Flagelin je sestavljen iz štirih domen, imenovanih D0-D3. Domeni D0 in D1 sta pomembni za aktivacijo TLR5 in sta med različnimi vrstami bakterij zelo ohranjeni, medtem ko sta domeni D2 in D3 pomembni za protitelesni odziv in predstavljata ti. hipervariabilno regijo. Namen diplomskega dela je bil izolirati in karakterizirati rekombinantni protein dimSF57, ki je sestavljen iz 2 kratkih flagelinov bakterije S. tryphiumrium povezanih med sabo s 57 aminokislinami dolgim peptidnim linkerjem. Kratek flagelin je sestavljen iz N-končnega dela flagelina (2-176 AK) in C-končnega dela flagelina (398-494 AK). Pri diplomskem delu smo uspešno izolirali in očistili rekombinanten flagelin z uporabo Ni-kelatne kromatografije in velikostno izključitvene kromatografije. Z metodo cirkularnega dikroizma smo pokazali, da je izoliran rekombinantni protein ustrezno zvit in zavzame sekundarno strukturo α-vijačnice. Z metodo dinamičnega sipanja svetlobe smo v raztopini vzorca pokazali delce velikosti 7,8 nm.

Keywords

TRL;TRL5;dimSF57;flagelin;rekombinantni proteini;diplomske naloge;

Data

Language:	Slovenian
Year of publishing:	2016
Typology:	2.11 - Undergraduate Thesis
Organization:	UM FKKT - Faculty of Chemistry and Chemical Engineering
Publisher:	[R. Bremšak]
UDC:	543.384:543.544.1(043.2)
COBISS:	20324886
Views:	1299
Downloads:	39
Average score:	0 (0 votes)
Metadata:

Other data

Secondary language:	English
Secondary title:	ISOLATION OF RECOMBINANT FLAGELLIN
Secondary abstract:	Toll-like receptors are type I transmembrane proteins characterized by an ectodomain, transmembrane domain and intracellular Toll/interleukin-1 receptor domain (TIR domain). TLRs recognize conserved structural motifs derived from pathogens known as pathogen-associated microbial patterns or PAMPs and endogenous danger signals that trigger immune response and production of proinflammatory cytokines. TLR5 recognizes flagellin from both Gram-positive and Gram-negative bacteria. Sensing of flagellin is evolutionary conserved mechanism. Flagellin is composed of four domains named D0 – D3. Domains D0 and D1 are highly conserved among bacteria and are important for activation of TLR5, whereas the hypervariable region D2 and D3 domains play important role in antibody response. The aim of this study was to isolate and characterize recombinant flagellin dimSF57 that is composed of 2 short flagellins linked together with 57 amino acids long peptide linker. Short flagellin is composed of N-terminal part (aa 2 – 176) and C-terminal part (aa 398 – 494) of fliC from bacteria Salmonella tryphiumrium. We have successfully isolated recombinant flagellin dim57SF by using Ni-NTA chromatography and were able to purify it with size exclusion cromathograpy. By using circular dichroism spectroscopy and dynamic light scattering we have determined the secondary structure and the size of dimSF57 being α-helical and 7,8 nm, respectively.
Secondary keywords:	flagellin;recombinant flagellin;TLRs;
URN:	URN:SI:UM:
Type (COBISS):	Bachelor thesis/paper
Thesis comment:	Univ. v Mariboru, Fak. za kemijo in kemijsko tehnologijo
Pages:	X, 37 str.
ID:	9168081