2.1. Expression and purification of the S glycoprotein and ACE2
SARS-CoV-2 S glycoprotein expression plasmids were constructed to encode
the ectodomain of S glycoprotein (residues 1–1208, with a mutated furin
cleavage site and two stabilizing proline substitutions K986P/V987P)
followed by a T4 foldon trimerization domain and a polyhistidine
purification tag, as described previously 34. For
functional assays, different
constructs were designed to encode S with mutations found in wild-type
human viruses or viruses from American mink and are referred to
throughout the text as SD614G,
SD614G+Y453F, and
SD614G+Δ69-70+Y453F. For structural analysis, a
SARS-CoV-2 S glycoprotein expression plasmid with four additional
stabilizing proline mutations (F817P, A892P, A899P,
A942P)35 was constructed and is referred to throughout
the text as mink S glycoprotein for simplicity.
ACE2 expression plasmids were also constructed as described previously
to encode the ectodomain of dimeric ACE2 followed by a human IgG1 Fc
purification tag, referred to throughout the text as hACE2-Fc and
mvACE2-Fc 34. Additionally, a monomeric mink ACE2
construct, where the IgG1 Fc purification tag was replaced with a
polyhistidine purification tag, was constructed for structure
determination and is referred to throughout the text as mvACE2. The
American mink ACE2 sequence was determined previously36.
Expi293F cells (Thermo Fisher, A14527) were cultured in Expi293
Expression Medium at 37°C and 8% CO2 in the Orbi-Shaker
(Sigma, Z763438) at 130 rpm. The cells were transiently transfected at a
3.0 x 106 cells/mL density using an Expifectamine 293
transfection kit (Thermofisher, A14524). For polyhistidine-tagged
proteins, the supernatant was harvested by centrifugation after 72 hours
and supplemented with 20 mM Imidazole, followed by affinity
chromatography with Ni2+-NTA agarose beads (GoldBio,
H-350). The sample was washed three times with 5 column volumes (CV) of
wash buffer (30 mM Na2HPO4 pH 7.4, 500
mM NaCl, 30 mM Imidazole) and then eluted three times with 5 CVs of
elution buffer (20 mM Na2HPO4 pH 7.4,
500 mM NaCl, 500 mM Imidazole). For Fc-tagged proteins, the supernatant
was harvested by centrifugation after 72 hours, followed by affinity
chromatography using HiTrap protein A columns (Cytiva). The sample was
washed with 10 CVs of wash buffer (20 mM sodium phosphate, pH 7.4) and
eluted with 5 CVs of elution buffer (0.1 M citric acid, pH 3). Sample
collection tubes were pre-loaded with neutralization buffer (1M Tris
HCl, pH 9).
The eluted proteins were concentrated with 50-kDa or 100-kDa MWCO
centrifugal filters. Protein samples used for bio-layer interferometry
(BLI) assays were used after affinity chromatography. Protein samples
used for structure determination were further purified by size exclusion
chromatography using the Superose 6 increase 10/300 GL column (Cytiva)
in buffer (20 mM Tris pH 8.0, 200 mM NaCl). The peak fractions were
collected and verified through SDS-PAGE gel analysis. The samples were
concentrated and flash-frozen in liquid nitrogen and stored at -80°C.