Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical
need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in
both clinical and basic science settings. To address this need, we have evaluated the
expression and purification of two previously reported S protein constructs in Expi293F
and ExpiCHO-S cells, two different cell lines selected for increased protein expression.
We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins.
Biochemical, biophysical, and structural (cryo-EM) characterizations of the SARS-CoV-2 S
proteins produced in both cell lines demonstrate that the reported purification strategy
yields high-quality S protein (nonaggregated, uniform material with appropriate
biochemical and biophysical properties), and analysis of 20 deposited S protein cryo-EM
structures reveals conformation plasticity in the region composed of amino acids
614–642 and 828–854. Importantly, we show that multiple preparations of
these two recombinant S proteins from either cell line exhibit identical behavior in two
different serology assays. We also evaluate the specificity of S protein-mediated host
cell binding by examining interactions with proposed binding partners in the human
secretome and report no novel binding partners and notably fail to validate the
Spike:CD147 interaction. In addition, the antigenicity of these proteins is demonstrated
by standard ELISAs and in a flexible protein microarray format. Collectively, we
establish an array of metrics for ensuring the production of high-quality S protein to
support clinical, biological, biochemical, structural, and mechanistic studies to combat
the global pandemic caused by SARS-CoV-2.
Coronavirus disease 2019 (COVID-19) is a global health crisis caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and there is a critical need to produce large quantities of high-quality SARS-CoV-2 Spike (S) protein for use in both clinical and basic science settings. To address this need, we have evaluated the expression and purification of two previously reported S protein constructs in Expi293F™ and ExpiCHO-S™ cells, two different cell lines selected for increased expression of secreted glycoproteins. We show that ExpiCHO-S™ cells produce enhanced yields of both SARS-CoV-2 S proteins. Biochemical, biophysical, and structural (cryo-EM) characterization of the SARS-CoV-2 S proteins produced in both cell lines demonstrate that the reported purification strategy yields high quality S protein (non-aggregated, uniform material with appropriate biochemical and biophysical properties). Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. We also evaluate the specificity of S protein-mediated host cell binding by examining interactions with proposed binding partners in the human secretome. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs, and in a flexible protein microarray format. Collectively, we establish an array of metrics for ensuring the production of high-quality S protein to support clinical, biological, biochemical, structural and mechanistic studies to combat the global pandemic caused by SARS-CoV-2.
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