Actinoporins (AP) are a group of α-pore-forming proteins found in the venom of sea anemones. In contact with target membranes, monomeric proteins oligomerize to form transmembrane pores through conformational change of the N-terminal α-helices. Typically, AP are known for their lack of cysteine residues and for binding to sphingomyelin, found in cell membranes. Their distinctive characteristics make them promising candidates for applications in biotechnology and medicine. Protein and genetic engineering have an important role in development of such applications, however there is also a fundamental demand for discovery and characterization of new AP and actinoporin-like proteins (ALP). In the scope of this thesis, we cloned a gene encoding protein CAND, ALP from Conus andremenezi, and recombinantly expressed it in Escherichia coli, purified and experimentally characterized for the first time. Amino acid sequence of CAND, as well as structural elements of secondary structure show numerous similarities to AP. CAND has melting temperature (Tm), around 41 °C (measured with CD spectroscopy), however salt concentration and pH have a significant effect on it. Increasing the salt concentration and lowering the pH have a positive effect on Tm. pH also showed significant effect on haemolytical activity of CAND, which decreases with lower pH values, and is in general few orders of magnitude lower compared to FraC. Like some of the ALP representatives that have been experimentally characterized to date, CAND does not show specificity for sphingomyelin, as it also binds to other lipids in the membrane. The pore forming activity of CAND protein was demonstrated by recording the pore of the CAND protein on large unilamilary vesicles (LUV) using cryo-electron microscopy. Pore isolation was performed by disruption of LUV with detergent DDM and subsequent separation with size exclusion chromatography.
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