*2.1. Obtaining of Recombinant Analog of Hct-S3 Actinoporin*

Hct-S3 (177 amino acid residues) is one of the most represented isoforms belonging to the multigene family of *H. crispa* actinoporins. A recombinant analog of Hct-S3 (rHct-S3) was expressed in *Escherichia coli* strain Rosetta (DE3) as fusion protein containing glutation-S-transpherase, polyhistidine tag, enteropeptidase cleavage site, and actinoporin. In order to avoid the denaturation of the target polypeptide and increase its yield, we applied a high-pressure homogenization approach using a French-press homogenizer for the cell destruction instead of ultrasonication. The fusion protein with a molecular mass of ~50 kDa was isolated using a metal-affinity chromatography and cleaved by enteropeptidase. Next, the targeted actinoporin was purified on a soybean trypsin inhibitor -affinity column and further desalted (Figure 1a). The final yield of rHct-S3 after high-pressure homogenization was 1 mg/L of cell culture in contrast to 0.2 mg/L yield after ultrasonication. The molecular mass of the polypeptide was determined by MALDI-TOF/TOF mass-spectrometry as 19,393 Da (Figure 1b), which is consistent with the predicted molecular mass (19,390 Da). The N-terminal amino acid sequence (15 aa) determined by the automated Edman degradation matched well with the amino acid sequence deduced from cDNA earlier.

**Figure 1.** Electrophoregram (**a**) and mass spectrum (**b**) of purified rHct-S3.
