Protein quality assessment
We assessed whether insect larvae-produced SARS CoV-2 S protein was
suitable for our intended use. The trimetric nature of the S protein,
which is typical of Type I viral fusion proteins, is a critical quality
attribute of recombinant S proteins in different expression systems
(Esposito et al., 2020). We evaluated the oligomeric state of the
IMAC-purified S protein from R. nu larvae by size exclusion
chromatography (SEC) in a Superdex 200 column. Figure 4a shows a typical
SEC chromatogram of the larvae-expressed SARS CoV-2 S protein eluting as
a broad peak with an elution volume close to 10 ml, compatible with a
450-500 kDa protein, in good accordance with the expected trimeric
nature of the purified protein. The S monomer is a protein with an
apparent molecular weight of ~ 150 kDa bearing many
glycan moieties, which affects its hydrodynamic behavior (the MW
calculated from the amino acid sequence is 138 kDa). The presence of S
protein in these fractions was confirmed by WB, using an anti 6xHis
tag-specific antibody (Figure 4b). Notably, two peaks, denoted as peak
#1 and #2, were also observed. However, these peaks were not
recognized by the anti 6xHis tag antibody in the WB revealed with ECL
(Figure 4b), indicating that they might correspond to non-related
impurities or to a dissociated and fragmented S protein with its
carboxyl-terminal cleaved. It should be noted that some proteolysis can
occur during concentration and buffer exchange of the sample performed
before injecting on a SEC column.