Serological testing is important for determining individuals who have been exposed to the virus, and the resulting antibody titer can facilitate identification of potential donors of convalescent plasma. Clinically, the production of large amounts of S ectodomain protein enables testing of individuals for SARS-CoV-2 seropositivity. (11,12)Дue to the global COVID-19 pandemic, the SARS-CoV-2 S ectodomain protein has become an important target for clinical, biological, and structural investigations, and future studies will require the efficient and streamlined production of this protein.

(8) In many coronaviruses, these events lead to conformational rearrangements in S2, which ultimately result in the fusion of the host and viral cell membranes, and delivery of the viral genome into the newly infected cell.

(8) Processing of both SARS-CoV and SARS-CoV-2 S proteins is dependent on the endosomal cysteine proteases cathepsin B and cathepsin L and the serine protease TMPRSS2. (8в€’10) Similar to SARS-CoV, the SARS-CoV-2 trimeric S glycoprotein mediates viral entry into the cell by utilizing angiotensin converting enzyme 2 (ACE2) as a human cell surface entry receptor. (8) The S protein in many Coronaviridae, including SARS-CoV-2, is cleaved between the S1 and S2 subunits (S1/S2 cleavage site) and at an additional site present in S2 (S2вЂ™ cleavage site). (4в€’7) Host cell proteases are essential for activating the S protein for Coronaviridae cellular entry. (4) The S1 subunit contains the receptor binding domain (RBD), which is responsible for host cell receptor binding, while the S2 subunit facilitates membrane fusion between the viral and host cell membranes.

The S protein is a class I viral fusion protein, which consists of two subunits (S1 and S2) and forms a trimer on the viral membrane. 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. In addition, the antigenicity of these proteins is demonstrated by standard ELISAs and in a flexible protein microarray format. 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. Importantly, we show that multiple preparations of these two recombinant S proteins from either cell line exhibit identical behavior in two different serology assays. 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 614вЂ“642 and 828вЂ“854.

We show that ExpiCHO-S cells produce enhanced yields of both SARS-CoV-2 S proteins. 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. 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.

Expi293 membrane pro