3.3 IL-18 DM1234 showed the lower aggregation capability with the higher activity
To evaluate the effect of the replacement of surface cysteine to serine on IL-18 aggregation, the oxidative stress of protein was induced by shaking the protein at 220 rpm in shaker incubator. The heat was also used to increase the energy of the reaction. After 16-18 h. of induction, the aggregated protein was then measured by ProteoStat protein aggregation assay. The result demonstrated that no difference in protein aggregation was observed between IL-18 DM and wild-type (Fig. 4A). However, IL-18 DM1234 showed significant decrease in protein aggregation determined by ProteoStat dye (Fig. 4A). This mutation could lower the aggregation ability of IL-18 DM1234 about 1.79 and 1.63 folds compared to IL-18 wild-type and IL-18 DM, respectively. Interestingly, when interferon-γ (IFN-γ) induction assay was performed with all three types of IL-18, the mutations of cysteine to serine showed the statistically increasing capability to stimulate the production of IFN-γ from NK-92MI cells (Fig. 4B). The levels of IFN-γ of IL-18 DM1234 indicated the higher efficacy approximately 10.0 and 2.8 folds compared to wild-type and DM IL-18, respectively. This suggested that the mutation of surface cysteine to serine enhanced the ability of IL-18 to activate NK-92MI in either way.