SDS-PAGE and Western blotting analysis of NK
SDS-PAGE analysis demonstrated that a 28 kDa protein was a crucial component in the supernatant from induced B. subtilis168/pHT01-apr N1, but that it was not present in the supernatant from B. subtilis 168/pHT01 and non-induced B. subtilis 168/pHT01-apr N1 fermentation broth (Fig. 2 A/B), suggesting that recombinant NK could be expressed in a soluble form. Western blotting with a His-tag-specific monoclonal antibody also showed a specific signal at 28 kDa, whereas no cross-reaction occurred in the total soluble proteins from non-induced B. subtilis 168/pHT01-apr N1 broth,which confirmed that the 28 kDa protein was the recombinant NK, as expected (Fig. 2 C).
In view of these reports, NK was produced by fed-batch cultures of recombinant B. subtilis , and its production was improved to 7,778 U/mL from 380 U/mL of flask culture using pH-stat and low-glycerol-level strategies. Future studies will design a process and set a kinetic model for fermentation optimization.