Expression and purification of the recombinant S protein
For recombinant proteins expression, HEK 293F cells were cultured at an
orbital shaker incubator in two 1 L flasks containing 500 mL culture
volume until the cell density reached 2 × 106cells/mL, and the cell survival rate was above 95%, then infected with
recombinant or wild-type baculoviruses (rB or wB) at a
multiplicity of infection (MOI) of 20 and incubated at 37 ℃. After 8 h,
sodium butyrate (Shanghai Macklin Biochemical Co., Ltd, China) was added
to the final concentration of 10 mM, and incubated for 3 days at 37 ℃ in
5% CO2. Cells were then collected, lysed in 1% Triton
X-100, and then centrifuged at 12000 × g for 30 min at 4 ℃. The
supernatants were prepared and examined with Western Blot as described
below.
The resulting precipitates were resuspended in 1 × phosphate buffer
saline (PBS) (pH = 7.4), ultrasonicated, and then centrifuged at 12000 ×g for 10 min at 4 ℃. The supernatants were collected and purified
using protein G REsin (GenScript, Nanjing, China) according to the
manufacturer’s instructions. The purified proteins were analyzed using
Coomassie Blue stained SDS-PAGE electrophoretogram, then quantified by
NanoDrop spectrophotometry (IMPLEN, Germany) and supplemented with 1%
protease inhibitor cocktail C (Beijing Yataihengxin Company, China) and
frozen at −80 °C until use.