Interakcija proteina egerolizina bakterije Spirosoma linguale z lipidnimi membranami

Hostnik, Maja (author), Butala, Matej (mentor)

Egerolizinska družina proteinov danes šteje preko 350 homologov, ki jih najdemo v številnih taksonih. Gre za majhne (13-20 kDa), β-strukturirane proteine z nizko izoelektrično točko in slabo poznano biološko vlogo. Egerolizini interagirajo z lipidnimi membranami in v kompleksu s proteinskim partnerjem tvorijo transmembranske pore. Egerolizini se komercialno uporabljajo za zatiranje rastlinskih škodljivcev, saj se specifično vežejo na lipid ceramid fosfoetanolamin v membranah nevretenčarjev. Gena za egerolizin in njegov hipotetični partnerski protein se nahajata tudi pri bakteriji Spirosoma linguale. Nedavno sta bila pripravljena plazmidna konstrukta za sintezo obeh proteinov, vendar je bil uspešno izoliran le rekombinantni egerolizin. V magistrski nalogi smo želeli pridobiti zadostno količino rekombinantnega egerolizina SlinAg in proteinskega partnerja SlinB bakterije Spirosoma linguale za analizo njune interakcije z umetnimi lipidnimi vezikli ter za analizo proti insekticidne aktivnosti izoliranih proteinov. Uspešno smo sintetizirali oba proteina v bakteriji Escherichia coli. Rekombinantni protein SlinAg smo pridobili z nikelj afinitetno kromatografijo iz topne frakcije in iz inkluzijskih telesc liziranega ekspresijskega seva. Rekombinantni protein SlinB smo izolirali iz inkluzijskih telesc in optimizirali njegovo renaturacijo. S testi vezave proteinov na umetne lipidne vezikle nismo ugotovili specifičnosti za posamezne lipide. Preliminarni testi toksičnosti kažejo, da bakterija Spirosoma linguale nima toksičnega učinka na ličinke koruznega hrošča.

• Language: Slovenian
• Keywords: egerolizin, membranski toksin, insekticid, Spirosoma linguale
• Work type: Master's thesis/paper
• Organization: BF - Biotechnical Faculty
• Year: 2019
• PID: 20.500.12556/RUL-107735
• COBISS.SI-ID: 5105999
• Publication date in RUL: 19.05.2019

Secondary language

• Language: English
• Title: Interaction of Spirosoma linguale aegerolysin protein with the lipid membranes

Abstract

Aegerolysin protein family comprises over 350 homologs from different taxonomic groups. They are small (13-20 kDa), β-structured proteins with low isoelectric point and poorly known biological role. Aegerolysins interact with lipid membranes and when complexed with a protein partner, form transmembrane pores. Aegerolysins are commercially used as insecticides due to their binding to ceramide phosphoethanolamine in invertebrate membranes. Genes for aegerolysin and its hypothetical protein partner are also found in bacteria Spirosoma linguale. Recombinant plasmids containing both genes, for the aegerolysin SlinAg and for its putative protein partner SlinB were recently constructed, however only the aegerolysin protein was purified. The aim of this master thesis was to acquire both recombinant proteins in concentrations high enough to analyse their interaction with artificial lipid vesicles and to analyse their potential insecticidal activity. Both recombinant proteins from bacteria Escherichia coli were successfully synthesized and purified. Recombinant protein SlinAg was purified by nickel affinity chromatography from the soluable fraction and from inclusion bodies. We purified recombinant protein SlinB from inclusion bodies and optimized its renaturation. Experiments with artificial vesicles did not show binding specificity for the selected lipids. Preliminary insecticidal tests showed that bacteria Spirosoma linguale is not toxic to western corn rootworm larvae.

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