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Priprava, izolacija in karakterizacija rekombinantnega proteina erilizina B
ID Mravinec, Martina (Author), ID Sepčić, Kristina (Mentor) More about this mentor... This link opens in a new window, ID Skočaj, Matej (Comentor)

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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.

Language:Slovenian
Keywords:egerolizini, proteini z domeno MACPF, porotvorni kompleks, ceramid fosfoetanolamin
Work type:Master's thesis/paper
Organization:BF - Biotechnical Faculty
Year:2020
PID:20.500.12556/RUL-120561 This link opens in a new window
COBISS.SI-ID:33522691 This link opens in a new window
Publication date in RUL:23.09.2020
Views:1718
Downloads:355
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Secondary language

Language:English
Title:Preparation, isolation and characterization of the recombinant protein erylysin B
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.

Keywords:aegerolysins, proteins with MACPF domain, pore-forming complex, ceramide phosphoethanolamine

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