Lysenin is a protein toxin from the earthworm Eisenia fetida which forms pores in lipid membranes. It is produced as a water-soluble monomer in cells, which binds to sphingomyelin containing lipid membranes. Upon binding, it forms nonameric pre-pores on the surface on membranes, which undergo large conformational changes in order to insert into membranes and form functional pores. Lysenin pore has unique structural, biochemical and biophysical properties and that is why lysenin pore is very interesting for use in different biotechnological applications. For these purposes, it is crucial to have efficient expression and purification system to produce sufficient amounts of pure monomeric recombinant protein. In this master thesis, we performed optimization of monomeric protein lysenin preparation, from plasmid construction to purification method as well as pore assembly. For purification method improvement, we designed a recombinant plasmid vector for bacterial expression of a protein with C-terminal polyhistidine tag, which enables affinity chromatography purification of lysenin. Change of polyhistidine tag position on lysenin from N- to C-terminus turned out as an improvement. Namely, in the case of polyhistidne tag at the C-terminus the yield was two times higher in comparison with N-terminally tagged protein. After efficient isolation and purification of monomeric lysenin we tested its pore forming activity and checked the correct folding via circular dichroism. Moreover, we assembled lysenin pores on model lipid membranes and isolated and purified them.
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