magistrsko delo
Povzetek
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.
Ključne besede
rekombinantni proteini;egerolizini;verzikolizin A;verzikolizin B;Trametes versicolor;
Podatki
Jezik: |
Slovenski jezik |
Leto izida: |
2023 |
Tipologija: |
2.09 - Magistrsko delo |
Organizacija: |
UL BF - Biotehniška fakulteta |
Založnik: |
[U. Kržič] |
UDK: |
579.255:577.112:577.352 |
COBISS: |
153696003
|
Št. ogledov: |
28 |
Št. prenosov: |
15 |
Ocena: |
0 (0 glasov) |
Metapodatki: |
|
Ostali podatki
Sekundarni jezik: |
Angleški jezik |
Sekundarni naslov: |
Isolation of recombinant proteins versicolysin A and B from bacteria Escherichia coli and their interaction with model lipid systems |
Sekundarni povzetek: |
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. |
Sekundarne ključne besede: |
recombinant proteins;aegerolysins;versicolysin A;versicolysin B;Trametes versicolor; |
Vrsta dela (COBISS): |
Magistrsko delo/naloga |
Študijski program: |
0 |
Konec prepovedi (OpenAIRE): |
1970-01-01 |
Komentar na gradivo: |
Univ. v Ljubljani, Biotehniška fak., Oddelek za mikrobiologijo |
Strani: |
1 spletni vir (1 datoteka PDF (XIV, 62 str., [4] f. pril.)) |
ID: |
19026793 |