Protein Purification.
Three mL of 1 M imidazole (Sigma-Aldrich) per 100 mL of transfected
clarified supernatant was added prior to purification. Purification used
two buffers, one binding buffer (20 mM sodium phosphate, 500 mM sodium
chloride, 30 mM imidazole pH 7.4) and one elution buffer (20 mM sodium
phosphate, 500 mM sodium chloride, 500 mM imidazole pH 7.4). The column
was first equilibrated with 5 column volumes (CV) of binding buffer. The
supernatant was then loaded onto an AKTA Avant 150 FPLC through a Cytiva
5 mL HisTrap FF column at 0.5 mL/min. After sample loading, the column
was washed with 5 CV binding buffer. The protein was eluted off the
column using a gradient elution from 100% binding buffer to 100%
elution buffer over 5 CV and peaks were collected in 2 mL fractions at
4°C. The peak fractions were loaded onto a 7K MWCO Zeba desalting column
at 4°C, buffer exchanged into 300 mM sodium chloride, 20 mM Tris-HCl pH
8, and later concentrated on an Amicon 100K MWCO Ultra-15 centrifugal
filter column at 4000 RCF at 4°C until desired concentration/volume was
met. A minimum of 500 μg of this affinity purified protein was then
loaded onto a Cytiva Superose 6 Increase 10/300 gel filtration column
also on the AKTA Avant 150 system. Preparative size exclusion
chromatography took place at 0.5 mL/min in 300 mM sodium chloride, 20 mM
Tris-HCl pH 8. Peaks corresponding to the size of spike trimer were then
fractioned into 2 mL fractions and later concentrated on a 100K MWCO
Amicon filter if needed. Protein quality was checked by SDS-PAGE gel and
analytical size exclusion chromatography before use.