*4.1. Expression and Isolation of Recombinant Hct-S3*

The recombinant plasmid obtained earlier was transformed into *E. coli* strain Rosetta (DE3) (Novagen, Merck KGaA, Darmstadt, Germany). Transformed cells were cultured at 37 ◦C in 1 L of Luria-Bertani medium containing 50 μg/mL kanamycin (Gibco, Thermo Fisher Scientific, Gaithersburg, MD, USA) until the optical density (OD600) ~0.5 was reached. After induction with IPTG at a concentration of 0.2 mM, the cells were incubated at 19 ◦C for 18 h at 180 rpm, centrifuged for 6 min at 6000 rpm at 4 ◦C, and supernatant was removed. The presence of rHct-S3 was determined in 12% polyacrylamide gel by Laemmli's SDS-PAGE method [43]. Precipitate was resuspended in the start buffer for affinity chromatography (400 mM NaCl, 20 mM Tris-HCl buffer, pH 8.0) and disrupted by French-press homogenizer (Thermo Fisher Scientific, Waltham, MA, USA) using the mini-cell (3.7 mL). Lysed cells were centrifuged for 10 min at 10,000 rpm to remove all insoluble particles. Supernatant was applied to a Ni-NTA agarose (Qiagen, Venlo, Netherlands), the fusion protein was purified with 5 volume of wash buffer (400 mM NaCl, 50 mM imidazole, 20 mM Tris-HCl buffer, pH 8.0) and 5 volume of start buffer. The fusion protein was cleaved by enteropeptidase (1 unit/mg protein) at room temperature for 18 h at 80 rpm. The recombinant actinoporin were purified from enteropeptidase on soybean trypsin inhibitor-agarose (Sigma-Aldrich, St. Louis, MO, USA) and desalted on a centrifugal filter tube (Millipore, Lenexa, KS, USA) with capacity < 3000 Da. The molecular masses of the purified rHct-S3 were analyzed by Ultra Flex III MALDI-TOF/TOF mass spectrometer (Bruker, Bremen, Germany). The amino acid sequence of rHct-S3 were determined on an automated sequencer protein Procise 492 Clc (Applied Biosystems, Foster City, CA, USA).

The purified rHct-S3 was dissolved in PBS, filtered by 0.22 μm "Millipore" membranes (Billerica, MA, USA) and used for the bioactivity experiments.
