magistrsko delo
Martina Mravinec (Author), Matej Butala (Reviewer), Kristina Sepčić (Mentor), Kristina Sepčić (Thesis defence commission member), Matej Butala (Thesis defence commission member), Uroš Petrovič (Thesis defence commission member), Matej Skočaj (Thesis defence commission member), Matej Skočaj (Co-mentor)

Abstract

Protein erilizin B (EryB) uvrščamo med proteine z domeno MACPF (Pfam PF01823). Sintetizira ga goba Pleurotus eryngii, njegova biološka vloga pa je še neznana. Njegov homolog je protein pleurotolizin B (PlyB), ki pa ga sintetizira goba Pleurotus ostreatus. Gobe iz tega rodu prav tako sintetizirajo proteine egerolizine, ki s proteini z domeno MACPF, tvorijo porotvorne komplekse. Dokazano je, da imajo egerolizini specifično afiniteto za vezavo na membrane, sestavljene iz kombinacij sfingomielina (SM) in holesterola ali pa ceramid fosfoetanolamina (CPE) in holesterola, proteini z domeno MACPF pa to vezavo ojačajo in skupaj z egerolizini tvorijo dvokomponentne transmembranske pore. V magistrski nalogi smo EryB sintetizirali v bakteriji Escerichia coli, ga uspešno izolirali in v kombinaciji z egerolizini iz gliv rodu Pleurotus (erilizin A, ostreolizin A6, pleurotolizin A2) preverili njegovo aktivnost z uporabo površinske plazmonske resonance, testom sproščanja kalceina in testom hemolize. Ugotovili smo, da EryB, podobno kot njegov homolog PlyB, tvori dvokomponentne komplekse z egerolizini in da so le ti porotvorni. Kompleksi egerolizin/EryB so manj uspešni pri tvorbi por, v primerjavi s kompleksi egerolizin/PlyB, za kar so najverjetneje odgovorni različni aminokislinski ostanki proteinov z domeno MACPF, ki prepoznajo egerolizine.

Keywords

egerolizini;proteini z domeno MACPF;porotvorni kompleks;ceramid fosfoetanolamin;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [M. Mravinec]
UDC: 577.2(043.2)
COBISS: 33522691 Link will open in a new window
Views: 690
Downloads: 297
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Other data

Secondary language: English
Secondary title: Preparation, isolation and characterization of the recombinant protein erylysin B
Secondary abstract: Protein erylysin B (EryB) is a protein with the MACPF domain (Pfam PF01823). It is produced by the mushroom Pleurotus eryngii. Its biological role is still unknown. Pleurotolysin B (PlyB), a homologue of EryB, is synthesized by Pleurotus ostreatus. Mushrooms of this genus also synthesize proteins called aegerolysins, which form pore-forming complexes with proteins that have MACPF domain. Aegerolysins have been shown to have a specific binding affinity for membranes, composed of sphingomyelin (SM) and cholesterol or ceramide phosphoetanolamine (CPE) and cholesterol. Proteins with MACPF domain further enhance this binding and form two-component transmembrane pores in concert with aegerolysins. As a part of this master's thesis, we expressed EryB in bacteria Escerichia coli. We successfully isolated it and tested its functionality in combination with Pleurotus aegerolysins erylysin A, ostreolysin A6, and pleurotolysin A2. Tests included surface plasmon resonance, calcein-release test and hemolysis test. We found that EryB, like its homologue PlyB, forms two-component complexes with aegerolysins and that these complexes are pore-forming. Aegerolysin/EryB complexes are less effective in pore formation than aegerolysin/PlyB complexes, most likely due to different aminoacids at some key sites, that are responsible for binding of proteins with MACPF domain to aegerolysins.
Secondary keywords: aegerolysins;proteins with MACPF domain;pore-forming complex;ceramide phosphoethanolamine;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. Ljubljana, Biotehniška fak.
Pages: XI, 62, [1] f.
ID: 12044669