Nanopore sensing is an analytical method that enables single molecule detection by identifying changes in ionic current that runs through the pore, as it interacts with the analyte. Due to an ever-increasing assortment of complex analytes the demand for pores with novel characteristics in growing as well. Herein we attempted to prepare homogeneous pores of perfringolysin O (PFO) for use in nanopore sensing. To this end we used sodium dodecyl sulphate agarose gel electrophoresis (SDS-AGE) to analyse the effects of different pore preparation conditions on size, homogeneity and final concentration of PFO oligomers, which we prepared on large unilamellar vesicles and solubilised with different detergents. Using ion exchange chromatography, SDS-AGE and size exclusion chromatography we then purified pore samples of pore preparation residues and simultaneously tried to resolve pores of varying shapes and sizes for further analysis on planar lipid membranes and with cryo transmission electron microscopy. Major requirement for adequate pore preparation was vesicle lipid composition, which had to ensure sufficient cholesterol exposure for binding of PFO to the membrane, while also being incapable of forming aggregates with PFO oligomers after vesicles were solubilised with detergent. Other tested conditions either had no effect on pore preparation or influenced it by affecting aggregation of oligomers with lipids after detergent addition. Using aforementioned methods, we successfully removed residues of pore formation from samples, but failed to resolve pores according to their sizes and shapes. Due to inability of purified pores to insert into lipid bilayers, we did not manage to characterise them on planar lipid membranes.
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