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12His‐SCOTIN (PRD) or 12His‐SCOTIN (PRDΔ150‐177) was overexpressed in E. coli BL21(DE3) using the pET28a vector. Bacterial cells were grown in Terrific Broth (TB) with vigorous shaking (200 rpm) at 37°C for 8 h and then incubated at 17°C for 36 h for protein expression. Bacterial pellets were resuspended in lysis buffer (50 mM Tris–HCl, 500 mM NaCl, 10% glycerol, and protease inhibitor cocktail; pH 7.9) and lysed by sonication. The lysate pellets were obtained by centrifugation at 12,000 g and 4°C for 30 min. Using a magnetic stirrer at RT for 2 h, the lysate pellets were dissolved in binding buffer (8 M urea, 50 mM Tris–HCl, 500 mM NaCl, 10% glycerol, and 25 mM imidazole; pH 7.9). Cell debris was removed as pellets by centrifuging lysate pellets in binding buffer at 12,000 g and 4°C for 30 min, and the supernatant was purified with a HisTrap™ HP column (Cytiva). The eluted proteins were further purified by gel filtration (HiLoad® 16/600 Superdex® 200 pg) using FPLC (ÄKTA go, Cytiva). The proteins were dialyzed with storage buffer (20 mM sodium acetate, 150 mM NaCl; pH 5) using desalting columns (PD‐10, Cytiva). Then, the proteins were concentrated to the concentration required for the experiment with Vivaspin® concentrators (Sartorius) and stored at −80°C.