magistrsko delo
Urška Kržič (Author), Kristina Sepčić (Reviewer), Matej Skočaj (Mentor)

Abstract

Verzikolizin A (VerA) je egerolizinski protein, ki ga proizvaja bazidiomiceta pisana ploskocevka (Trametes versicolor). Prav tako proizvaja tudi partnerski protein z domeno, ki napade membrano/perforin (MACPF), imenovan verzikolizin B (VerB). Rekombinantna proteina VerA in VerB smo izrazili v bakteriji Escherichia coli ter ju proizvedli v rekombinantni obliki. Prav tako smo pripravili delecijsko mutanto VerA (Δ37VerA), brez začetnih 37 aminokislinskih ostankov. Za vse proteine smo pripravili napoved tridimenzionalne strukture proteina. S testom sedimentacije smo v nadaljevanju preverjali, ali VerA in Δ37VerA prepoznata specifične membranske lipide ali lipidne mešanice. Pri tem smo ugotovili, da je VerA nestabilen protein, ki se obarja. Za Δ37VerA smo ugotovili, da se veže na lipidne vezikle iz totalnega lipidnega ekstrakta žuželčje celične linije Sf9. V nadaljevanju smo preverjali, ali mešanica VerA in VerB tvori citolitične komplekse. Pri tem smo uporabili test hemolize na govejih eritrocitih ter ugotovili, da je mešanica Δ37VerA in VerB hemolitična. Hemolitična aktivnost pa je bila boljša v kislih pogojih. Citolitično aktivnost smo preizkušali tudi na žuželčji celični liniji Sf9 in vitro, kjer smo ugotovili, da citolitični kompleksi VerA/VerB ali Δ37VerA/VerB ne delujejo citolitično na te celice.

Keywords

rekombinantni proteini;egerolizini;verzikolizin A;verzikolizin B;Trametes versicolor;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [U. Kržič]
UDC: 579.255:577.112:577.352
COBISS: 153696003 Link will open in a new window
Views: 28
Downloads: 15
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Other data

Secondary language: English
Secondary title: Isolation of recombinant proteins versicolysin A and B from bacteria Escherichia coli and their interaction with model lipid systems
Secondary abstract: Versicolysin A (VerA) is an aegerolysin protein produced by basidiomycetes Trametes versicolor. This mushroom also produces a partner protein versicolysin B (VerB) with membrane attack complex/perforin (MACPF) domain. The purpose of this thesis was to prepare and express recombinant proteins VerA and VerB, and to characterize their biochemical properties. Recombinant proteins were expressed and produced in bacterium Escherichia coli. We also obtained a VerA deletion mutant (Δ37VerA) without the first 37 amino acids. These amino acids are usually not present in other known aegerolysins. Furthermore, we predicted the 3D structures for all these three proteins. Using the sedimentation test, we showed that VerA is not a stable protein and that it precipitates in solution. On the other hand, we found that Δ37VerA binds to lipid vesicles reconstituted from the insect cell line Sf9 total lipid extract. While the combination of VerA and VerB did not induce hemolysis, the combination of relatively high concentrations of Δ37VerA and VerB did show hemolytic activity and this activity was enhanced in acidic conditions. We also tested VerA/VerB or Δ37VerA/VerB cytolytic activity on the insect cell line Sf9 in vitro and found that these protein combinations are not cytolytic for these cells.
Secondary keywords: recombinant proteins;aegerolysins;versicolysin A;versicolysin B;Trametes versicolor;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 1970-01-01
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo
Pages: 1 spletni vir (1 datoteka PDF (XIV, 62 str., [4] f. pril.))
ID: 19026793