3.2 Expression and refolding of recombinant proteins
The expression of each type of IL-18 was performed in E. coliRosetta2 (DE3) pLysS. After cell lysis by using sonication method, both soluble and inclusion body of crude protein were analyzed by SDS-PAGE. Figure 2 demonstrated that a protein band about 20 kDa corresponding to mature form of human IL-18 was detected in both soluble fraction and inclusion body extracted from E. coli harboring pET28a containing IL-18 ORF insert while the same molecular weight was not observed in transformant having an empty vector (data not shown). However, more than 80% of this protein was found as inclusion form in all type of IL-18 (Fig. 2A lane 4, 5 and 6). This result suggested that recombinant human IL-18 was successful expressed in E. coli mostly as inclusion body. Therefore, in order to refold the protein to correct the structure of IL-18, step-wised refolding methods were then performed after washing steps. The result showed that the purity of the protein after refolding step was greater than 95% in SDS-PAGE analysis with yield less than the beginning source (Fig. 2B). Western blotting was also used to demonstrate the specific protein bands by anti-human IL-18 antibody (Fig. 2C). We validated the recombinant IL-18 by excising the protein band from the gel and subjecting to LC-MS/MS analysis. Tryptic digestion of this protein provided 9 fragments of peptides covered about 48.0% of pro-form of IL-18 or 58.8% of mature form IL-18 (Fig. 3A and 3B). These data suggested that this refolded IL-18 was mature IL-18.