2.2 Stepwise refolding
The pellet from sonication step was washed by 25 ml PBS 3 time and centrifuged at 10,000 rpm for 10 min. Pellet was then collected, weighted and resuspended in solubilization buffer [base buffer (50 mM Tris-HCl, 150 mM NaCl, 100 mM Glycine, 20% Glycerol, pH 8.0) containing 8 M urea] with the final concentration of 2-8 mg/ml. The suspension was then mixed in orbital shaker for 30 min and centrifuged at 15,000 rpm for 15 min before filtrating with 0.45 μm syringe filter. The supernatant was then dialyzed with slide-A-Lyzer G2 dialysis cassettes (Thermo Scientific, USA) toward 1 L of base buffer containing 6M urea for 4 h at 4°C. During this step, 500 mL of base buffer was added to dialysis buffer every 6 or 12 h for 4 times. Finally, the dialysis cassette with solubilized inclusion body was placed in 2 mL purely base buffer for 6 h. The refolded IL-18 were then harvested and kept at 4 °C until use.