Izolacija in lastnosti rekombinantinh proteinov erilizina B in pleurotolizina B, pripravljenih v kvasovki Pichia pastoris

Popošek, Larisa Lara (avtor), Skočaj, Matej (mentor), Grundner, Maja (komentor)

Proteina erilizin B (EryB) in pleurotolizin B (PlyB) sta 59 kDa velika proteina iz proteinske družine kompleksa, ki napade membrano/perforina (MACPF). Erilizin B je protein iz užitne gobe kraljevi ostrigar (Pleurotus eryngii), pleurotolizin B pa iz užitne gobe bukov ostrigar (P. ostreatus). V kombinaciji z egerolizinskim proteinskim partnerjem tvorita multimerne bikomponentne transmembranske pore v membranah, ki vsebujejo specifične lipidne receptorje, kar vodi v lizo tarčnih celic. Te proteine lahko rekombinantno izražamo v bakteriji Escherichia coli, vendar se proteina ne zvijata pravilno in se skoncentrirata v inkluzijskih telescih, zaradi česar je potrebna izolacija telesc in dodatno zvijanje proteina. Proteina EryB in PlyB smo zato želeli izražati v kvasovki Pichia pastoris ob uporabi konstitutivno izraženega promotorja GAP in z uporabo ekspresijskega vektorja pGAPZαA. Pričakovali smo, da se bosta proteina izražala v topni obliki in ju bomo na koncu lahko izolirali v višjih koncentracijah. Izražanje obeh proteinov smo preverjali z različnimi metodami. Spremljali smo hitrost rasti kvasovke P. pastoris in proteina skušali zaznati s poliakrilamidno gelsko elektroforezo ter s prenosom western, vendar proteinov nismo zaznali. Erilizin B in PlyB sta v kombinaciji z egerolizinskim partnerjem ostreolizinom A6 hemolitična, zato smo njuno prisotnost v gojišču preverili tudi s testom hemolize, vendar do lize eritrocitov ni prišlo. Na podlagi vseh rezultatov zato sklepamo, da kvasovka P. pastoris z ekspresijskim vektorjem pGAPZαA ni primeren ekspresijski sistem za pridobivanje rekombinantnih proteinov EryB in PlyB.

• Jezik: Slovenski jezik
• Ključne besede: rekombinantni proteini, proteini z domeno MACPF, erilizin B, pleurotolizin B, izolacija, kvasovke, Pichia pastoris
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: BF - Biotehniška fakulteta
• Kraj izida: Ljubljana
• Založnik: [L. L. Popošek]
• Leto izida: 2021
• PID: 20.500.12556/RUL-130257
• UDK: 577.112:582.28
• COBISS.SI-ID: 76657923
• Datum objave v RUL: 12.09.2021

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Isolation and characterization of recombinant proteins erylysin B and pleurotolysin B, produced in yeast Pichia pastoris

Izvleček

Proteins erylysin B (EryB) from king oyster mushroom (Pleurotus eryngii) and pleurotolysin B (PlyB) from oyster mushroom (P. ostreatus) are 59 kDa proteins belonging to MACPF protein superfamily. The combination of these proteins and an appropriate aegerolysin protein partner forms bi-component multimeric transmembrane pore in membranes with specific lipid receptors. Erylysin B and PlyB can be expressed recombinantly in bacteria Escherichia coli, but they do not fold properly and are found in inclusion bodies. Therefore, the inclusion bodies need to be isolated and the proteins refolded to gain active proteins. The goal of our research was to express soluble EryB and PlyB in the yeast Pichia pastoris in higher concentrations. We monitored the expression of recombinant proteins EryB and PlyB with several tests, including gel electrophoresis and Western blot. Erylysin B and PlyB are lytic in the presence of aegerolysin protein partner ostreolysin A6, therefore the presence of proteins in the media was tested by monitoring the hemolysis.The lysis of erythrocytes did not occur, so we concluded that the yeast P. pastoris in the combination with pGAPZαA expression vector is not a suitable expression system for the production of recombinant EryB and PlyB.

• Ključne besede: recombinant proteins, MACPF proteins, erylysin B, pleurotolysin B, isolation, yeasts, Pichia pastoris