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.