Figure legends
Figure 1. The crystal structure of IL-18 WT and receptor. (A) The ribbon structure of mature human IL-18 (3WO3) retrieved from Protein Data Bank. Surface cysteines substituted in this study were indicated. (B) The ribbon structure of IL-18 (cyan) with IL-18 receptor α (yellow).
Figure 2. SDS-PAGE analysis of recombinant IL-18 before and after refolding. (A) SDS-PAGE analysis of soluble protein and insoluble protein obtained after IL-18 expression by E. coli . Lane: M = protein marker, 1 = soluble protein from IL-18 WT expressing E. coli , 2 = soluble protein from IL-18 DM expressing E. coli , 3 = soluble protein from IL-18 DM1234 expressing E. coli , 4 = insoluble protein from IL-18 WT expressing E. coli , 5 = insoluble protein from IL-18 DM expressing E. coli , 6 = insoluble protein from IL-18 DM1234 expressing E. coli . (B) SDS-PAGE analysis of recombinant IL-18 after protein refolding. M = protein marker, 1 = IL-18 WT, 2 = IL-18 DM, 3 = IL-18 DM1234. All samples were loaded onto 12% SDS-PAGE.
Figure 3. LC-MS/MS analysis of recombinant mature IL-18. (A) Tryptic peptide map of recombinant mature IL-18 produced by E. coli expression system. The identified peptides are shown in red, (B) Identified tryptic peptide fragment derived from IL-18.
Figure 4. Aggregation and IFN-γ induction assays of each type of IL-18. (A) The signal obtained from ProteoStat protein aggregation assay of each type of refolded IL-18 was shown. (B) IFN-γ induction assay with each type of IL-18. NK-92MI cells were treated with various concentrations of IL-18 for 16-18 hours. IFN-γ was measured in the supernatant by ELISA. All figures illustrated results represent the mean ± SD of three independent experiments. *p < 0.05.
Figure 5. Structural analysis revealed by MD simulation. (A) Structural alignment of three types of IL-18. The binding site I is indicated in the figure. (B) The obvious different part of IL-18 DM1234 structure. The interface between binding site I of each type of IL-18 and IL-18 receptor α was demonstrated. (C) The molecular structure revealed the interaction between D35 of IL-18 WT and IL-18 DM1234 and V125 and S127 of IL-18 receptor α. The distance between interacted amino acids was indicated as angstrom (A˚). (D) The interaction of D35 of IL-18 DM and V125 and S127 of IL-18 receptor α was demonstrated with the distance as angstrom (A˚).