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Priprava rekombinantnih proteinov heterolizina A in B v bakteriji Escherichia coli in njuna interakcija z umetnimi lipidnimi membranami
ID Hrovatin, Matija (Author), ID Skočaj, Matej (Mentor) More about this mentor... This link opens in a new window

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Abstract
Egerolizini so pretežno β-strukturirani proteini, z molekulsko maso 15-20 kDa, ki prepoznajo in se vežejo na različne lipide oziroma lipidne mešanice. Nekateri egerolizini skupaj s partnerskimi proteini, ki vsebuje domeno, ki napade membrano/perforin (MACPF), tvorijo citolitične komplekse in povzročijo nastanek multimernih transmembranskih por v tarčnih celicah. S pomočjo bioinformatskih analiz smo ugotovili, da tudi bazidiomiceta ameriška rdeča trohnoba (Heterobasidion irregulare) vsebuje egerolizin in protein z domeno MACPF. Egerolizin smo poimenovali heterolizin A (HetA), protein z domeno MACPF pa heterolizin B (HetB). Namen magistrske naloge je bil (i) priprava rekombinantnih proteinov HetA in HetB v bakteriji Escherichia coli, (ii) opis njunih biokemijskih lastnosti, (iii) ovrednotiti, ali se HetA veže na biološke membrane ter (iv) preverjanje njune domnevne porotvorne aktivnost. Medtem ko smo rekombinantni HetA uspeli pripraviti, nam priprava proteina HetB ni uspela. To je bil tudi razlog, da smo za preverjanje porotvorne aktivnosti uporabili proteina pleurotolizin B (PlyB) in pulmolizin B (PulB), ki sta homologa HetB. S pomočjo turbidimetričnega hemolitičnega testa na govejih eritrocitih, s testom zasledovanja sproščanja fluorescenčnega barvila kalceina iz umetnih lipidnih veziklov, ter s testom citotoksičnosti na žuželčji celični linije Sf9 in vitro smo potrdili nastanek porotvornih citolitičnih kompleksov HetA/PlyB in HetA/PulB. Z uporabo površinske plazmonske resonance in sedimentacijskih testov smo dokazali vezavo egerolizina HetA na ceramid fosfoetanolamin in sfingomielin. Zaključimo lahko, da egerolizin HetA v kombinaciji s PlyB ali PulB, izkazuje citolitični potencial, kar bi lahko bilo potencialno uporabno v smislu zaščite rastlin ali v biomedicini.

Language:Slovenian
Keywords:Egerolizini, proteini z domeno MACPF, heterolizin A, heterolizin B, Heterobasidion irregulare, umetne lipidne membrane, rekombinantni proteini
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2023
PID:20.500.12556/RUL-147084 This link opens in a new window
COBISS.SI-ID:157137923 This link opens in a new window
Publication date in RUL:23.06.2023
Views:756
Downloads:79
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Secondary language

Language:English
Title:Production of recombinant proteins heterolysin A and heterolysin B from bacterium Escherichia coli and their interaction with model lipid membranes
Abstract:
Aegerolysins are small (15-20 kDa), β-structured proteins that recognize and bind to various lipids or lipid mixtures. It has been shown that aegerolysins, together with a partner protein that contains a membrane-attack/perforin domain (MACPF), form cytolytic complexes and cause the formation of multimeric transmembrane pores in target cell membranes. Using bioinformatic analyses, we have found that the basidiomycete Heterobasidion irregulare codes for aegerolysin and a protein partner with MACPF domain. We have named the aegerolysin heterolysin A (HetA) and protein with MACPF domain heterolysin B (HetB). The aim of this master thesis was to (i) recombinantly express the proteins HetA and HetB in bacterium Escherichia coli, (ii) describe biochemical properties of both proteins, (iii) test their pore-forming ability and (iv) inspect whether HetA binds to biological membranes. While the expression of recombinant HetA was successful, we were unable to express protein HetB. Because the recombinant production of HetB failed, we used its homologues, pleurotolysin B (PlyB) and/or pulmolysin B (PulB) in the further studies. Using a turbidimetric hemolytic assay on bovine erythrocytes, monitoring the release of the fluorescent dye calcein from artificial lipid vesicles, and performing a cytotoxicity assay on Sf9 insect cells, we confirmed the formation of HetA/PlyB and HetA/PulB pore-forming cytolytic complexes. Furthermore, we have demonstrated the binding of aegerolysin HetA to ceramide phosphoethanolamine and sphingomyelin using surface plasmon resonance and sedimentation tests. We can conclude that HetA in combination with appropriate MACPF partner protein shows cytolytic potential, which could be used in medical research and in agriculture for pest control.

Keywords:Aegerolysins, proteins with MACPF domain, heterolysin A, heterolysin B, Heterobasidion irregulare, model lipid membranes, recombinant proteins

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