Aegerolysins are small (15-20 kDa), β-structured proteins that recognize and bind to various lipids or lipid mixtures. It has been shown that aegerolysins, together with a partner protein that contains a membrane-attack/perforin domain (MACPF), form cytolytic complexes and cause the formation of multimeric transmembrane pores in target cell membranes. Using bioinformatic analyses, we have found that the basidiomycete Heterobasidion irregulare codes for aegerolysin and a protein partner with MACPF domain. We have named the aegerolysin heterolysin A (HetA) and protein with MACPF domain heterolysin B (HetB). The aim of this master thesis was to (i) recombinantly express the proteins HetA and HetB in bacterium Escherichia coli, (ii) describe biochemical properties of both proteins, (iii) test their pore-forming ability and (iv) inspect whether HetA binds to biological membranes. While the expression of recombinant HetA was successful, we were unable to express protein HetB. Because the recombinant production of HetB failed, we used its homologues, pleurotolysin B (PlyB) and/or pulmolysin B (PulB) in the further studies. Using a turbidimetric hemolytic assay on bovine erythrocytes, monitoring the release of the fluorescent dye calcein from artificial lipid vesicles, and performing a cytotoxicity assay on Sf9 insect cells, we confirmed the formation of HetA/PlyB and HetA/PulB pore-forming cytolytic complexes. Furthermore, we have demonstrated the binding of aegerolysin HetA to ceramide phosphoethanolamine and sphingomyelin using surface plasmon resonance and sedimentation tests. We can conclude that HetA in combination with appropriate MACPF partner protein shows cytolytic potential, which could be used in medical research and in agriculture for pest control.
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