2.1 Plasmid construction and protein expression
All plasmids were synthesized and obtained from GenScript company (NJ, USA). Open reading frame (ORF) of IL-18 with optimized codon forE. coli expression system was inserted between Nde l andEco RI restriction sites of pET28a expression vector. The mutant IL-18 including IL-18 E6K+T63A double mutation (IL-18 DM) and IL-18 E6K+T63A+C38S+C68S+C76S+C127S (IL-18 DM1234) were also obtained from the same source with similar restriction sites and vector. All recombinant plasmids (pET28a-IL-18WT, pET28a-IL-18DM and pET28a-IL-18DM1234) were transformed to E. coli Rosetta2 (DE3) pLysS and selected by Luria Bertani (LB) agar (Sigma-Aldrich, USA) containing 34 mg/ml of chloramphenicol (Invitrogen, USA) and 50 mg/ml of kanamycin (Invitrogen, USA). The selected clones were then used for IL-18 production. Briefly, each type of IL-18 bearingE. coli was grown in LB broth containing 34 mg/ml of chloramphenicol and 50 mg/ml of kanamycin and incubated in shaker incubator (Wiggens, Germany) at 37 °C, 200 rpm till OD600 reach 0.6. After that, 1 M IPTG (Sigma-Aldrich, USA) was then added to the culture with the final concentration of 0.1 M and incubated at 25 °C in shaker incubator with 200 rpm for 5 h. Bacterial cells were then harvested by centrifugation at 10,000 rpm for 10 min, resuspended in 10 ml PBS and sonicated on ice with 60% amplitude and pulses of 10s: 15s for 5 min. Cell lysate was then centrifuged for 10 mins at 10,000 rpm at 4 °C. The supernatant and cell pellet were separated and checked by 12% gel SDS-PAGE.