Lastnosti egerolizina bakterije Spirosoma linguale

Snoj, Jaka (author), Butala, Matej (mentor)

Egerolizini so družina majhnih (13-20 kDa) β-strukturiranih proteinov, ki jih najdemo pri predstavnikih evkariontov in prokariontov. Večina egerolizinov interagira z membranskimi lipidi, pri določenih pa so odkrili lastnost porotvornosti v interakciji s proteinskim partnerjem. Bakterije, ki imajo v operonu poleg gena za egerolizin še gen za protein, ki intreragira z egerolizinom, izkazujejo insekticidne lastnosti. Ena izmed bakterij, pri kateri so zasledili operon z genom za egerolizin in z genom za protein, ki domnevno tvori insekticidni kompleks, je bakterija Spirosoma linguale. Namen mojega magistrskega dela je bil pridobiti rekombinantni egerolizin SlinAg in protein SlinB, ki imata gena na skupnem operonu bakterije S. linguale. Izolirana proteina nam omogočita karakterizacijo interakcije teh dveh proteinov, med seboj in v interakciji z lipidnimi membranami. Iz genomske DNA bakterije S. linguale smo pomnožili gen za egerolizin SlinAg in gen za proteinski partner SlinB. Proteina smo izrazili v heterolognem ekspresijskem sistemu v bakteriji Escherichia coli. Z nikljevo afinitetno kromatografijo smo izolirali egerolizin v topni obliki. Z uporabo cirkularnega dikroizma smo določili sestavo sekundarne strukture rekombinantnega egerolizina in dokazali predominantno β-strukturiranost proteina. Proteina SlinB nismo pridobili v topni obliki, zato nismo preverili v kolikor proteina tvorita kompleks na membrani.

• Language: Slovenian
• Keywords: egerolizin, membranski toksin, insekticid, Spirosoma linguale
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2018
• PID: 20.500.12556/RUL-102830
• COBISS.SI-ID: 4895567
• Publication date in RUL: 08.09.2018

Secondary language

• Language: Unknown
• Title: Characteristics of Spirosoma linguale aegerolysin

Abstract

Aegerolysin protein family comprises small (13-20 kDa) and mostly β-structured proteins that are present in organisms from different kingdoms (bacteria, eukaryotes). Some aegerolysins interact with membranes and when complexed with a protein partner exhibit pore-forming abilities. In bacteria, egerolysin and its protein partner are encoded on an operon and this bacterial egerolysin complexes exert insecticidal characteristics. The genome of a soil bacterium Spirosoma linguale carries such an operon encompassing an aegerolysin and a putative protein partner genes. The aim of this master theses was to isolate recombinant S. linguale aegerolysin and its protein partner and to characterize the interaction between the two proteins and the interaction of the proteins with the lipid membrane. We successfully constructed recombinant plasmids containing both genes, for the aegerolysin SlinAg and for its putative protein partner SlinB, however we managed to purify only the aegerolysin protein. We characterized the SlinAg protein by circular dichroism spectroscopy which showed that SlinAg is composed of mainly β-strands, which is typical characteristic of aegerolysins. Since we could not isolate SlinB we were not able to assay if the two proteins form a complex on the membrane.

• Keywords: aegerolysin, membrane toxin, insecticide, Spirosoma linguale