Actinoporins and actinoporin-like proteins represent a large group of α-pore-forming proteins. Upon binding to membranes, a conformational change of the monomer form occurs, followed by oligomerization, where a larger number of monomers assemble to form a pore. Lots of research and development is being conducted to be able to use pores as biosensors. Such application requires a robust pore, which can be further modified to augment its properties. As part of the Master's thesis, we expressed 10 actinoporin homolog constructs based on the EST sequence from Orbicella faveolata (Fav) in E.coli BL21 (DE3) cells. Proteins were isolated and purified by nickel-affinity chromatography, and their pore-forming ability was confirmed by performing a hemolysis test. The ability to oligomerize and insert into lipid membranes was also confirmed on the Orbit mini system, where we successfully inserted all constructs into the DOPC:SM membrane. We were able to perform biophysical characterization. To select the construct that exhibits the smallest electric current noise compared to the control construct, we performed preliminary pore formation and analysis with the Orbit mini system on more stable membranes made of DPhPC lipids. The construct with the lowest noise was used for histone detection experiments. For successful detection, it was necessary to optimize the electric voltage, its polarity, buffer solutions and protein concentration, so that despite Fav’s high tendency to insert into the membranes, we obtained measurements with a low current, which reflects the presence of only one or two pores. Upon the addition of histones H3.1 and H4, different types of blockages were observed, which indicates the ability to separate between the two histone variants, which mainly differ only in the net charge and the length of the N-terminal positively charged histone tail.
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