Within this thesis, we aimed to obtain the soluble recombinant protein beauveriolysin B (BlyB) in the bacterium Escherichia coli. In addition, we sought to determine whether the protein pair beauveriolysin A (BlyA)/BlyB forms transmembrane pores, thereby defining their biological role. For the biochemical characterization of BlyB, a recombinant plasmid vector encoding a deletion mutant of BlyB (Δ25BlyB) was constructed. Plasmid vectors encoding BlyA and BlyB were transformed into competent E. coli BL21(DE3) cells, and transformants were obtained in both cases. The BlyA protein was successfully isolated and purified using Ni-affinity chromatography, and its lipid specificity was analyzed by surface plasmon resonance. We confirmed previous findings that the aegerolysin BlyA binds strongly to liposomes containing sphingomyelin/cholesterol (SM:Chol) lipid complexes, but not to liposomes containing ceramide phosphoethanolamine/cholesterol/phosphatidylcholine (CPE:PC:Chol). Despite multiple attempts, the recombinant protein Δ25BlyB could not be obtained. Possible reasons for this failure include the toxicity of the protein to E. coli cells or the instability of the protein or its mRNA.
|
