Recombinant SARS-CoV-2 A222V Spike S1 Subunit His Protein CF

Name: Recombinant SARS-CoV-2 A222V Spike S1 Subunit His Protein CF
Brand: R&D Systems
SKU: 10725-CV

Catalog # 10725-CV

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Key Product Details

Source:	HEK293
Accession #:	YP_009724390.1
Applications:	Bioactivity

View Full Technical Details for Recombinant SARS-CoV-2 A222V Spike S1 Subunit His Protein CF

Product Specifications

Source	Human embryonic kidney cell, HEK293-derived sars-cov-2 Spike S1 Subunit protein Val16-Pro681 (A222V), with a C-terminal 6-His tag
Purity	>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin level	<0.10 EU per 1 μg of the protein by the LAL method.
N-terminal sequence Analysis	Val16
Predicted Molecular Mass	75 kDa
SDS-PAGE	108-122 kDa, under reducing conditions
Activity	Measured by its binding ability in a functional ELISA with Recombinant Human ACE-2 His-tag (Catalog # 933-ZN).

Recombinant SARS-CoV-2 A222V Spike S1 Subunit His Protein CF Scientific Data Examples

	Recombinant SARS-CoV-2 A222V Spike S1 Subunit His-tag Protein Binding Activity. Recombinant SARS-CoV-2 A222V Spike S1 Subunit His-tag (Catalog # 10725-CV) binds Recombinant Human ACE-2 His-tag (933-ZN) in a functional ELISA.
	Recombinant SARS-CoV-2 A222V Spike S1 Subunit His-tag Protein SDS-PAGE. 2 μg/lane of Recombinant SARS-CoV-2 A222V Spike S1 Subunit His-tag (Catalog # 10725-CV) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 108-122 kDa.
	Binding of ACE-2 to SARS-CoV-2 A222V mutant Spike S1 subunit protein by surface plasmon resonance (SPR). Recombinant SARS-CoV-2 Spike S1 subunit A222V His-tag was immobilized on a Biacore Sensor Chip CM5, and binding to recombinant human ACE-2 (933-ZN) was measured at a concentration range between 0.092 nM and 47.2 nM. The double-referenced sensorgram was fit to a 1:1 binding model to determine the binding kinetics and affinity, with an affinity constant of KD=1.815 nM. (Biacore T200).

Formulation, Preparation and Storage

Carrier Free

What does CF mean?

CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.

What formulation is right for me?

In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.

10725-CV

Formulation	Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose.
Reconstitution	Reconstitute at 500 μg/mL in PBS.
Shipping	The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	Use a manual defrost freezer and avoid repeated freeze-thaw cycles. 12 months from date of receipt, -20 to -70 °C as supplied. 1 month, 2 to 8 °C under sterile conditions after reconstitution. 3 months, -20 to -70 °C under sterile conditions after reconstitution.

Reconstitution Calculator

The reconstitution calculator allows you to quickly calculate the volume of a reagent to reconstitute your vial. Simply enter the mass of reagent and the target concentration and the calculator will determine the rest.

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Background: Spike S1 Subunit

SARS-CoV-2, which causes the global pandemic coronavirus disease 2019 (Covid-19), belongs to a family of viruses known as coronaviruses that are commonly comprised of four structural proteins: Spike protein(S), Envelope protein (E), Membrane protein (M), and Nucleocapsid protein (N) (1). SARS-CoV-2 Spike Protein (S Protein) is a homotrimeric glycoprotein that mediates membrane fusion and viral entry. As with most coronaviruses, proteolytic cleavage of the SARS-CoV-2 S protein into two distinct peptides, S1 and S2 subunits, is required for activation. The S1 subunit is focused on attachment of the protein to the host receptor while the S2 subunit is involved with cell fusion (2-5). A SARS-CoV-2 variant carrying the S protein amino acid (aa) change A222V has been rapidly spreading across Europe (6,7). Based on structural biology studies, the receptor binding domain (RBD), located in the C-terminal region of S1, can be oriented either in the up/standing or down/lying state (8). The standing state is associated with higher pathogenicity and both SARS-CoV-1 and MERS can access this state due to the flexibility in their respective RBDs. A similar two-state structure and flexibility is found in the SARS-CoV-2 RBD (9). Based on amino acid (aa) sequence homology, the SARS-CoV-2 S1 subunit has 65% identity with SARS-CoV-1 S1 subunit, but only 22% homology with the MERS S1 subunit. The low aa sequence homology is consistent with the finding that SARS and MERS bind different cellular receptors (10). The S Protein of the SARS-CoV-2 virus, like the SARS-CoV-1 counterpart, binds Angiotensin-Converting Enzyme 2 (ACE-2), but with much higher affinity and faster binding kinetics (11). Before binding to the ACE-2 receptor, structural analysis of the S1 trimer shows that only one of the three RBD domains in the trimeric structure is in the "up" conformation. This is an unstable and transient state that passes between trimeric subunits but is nevertheless an exposed state to be targeted for neutralizing antibody therapy (12). Polyclonal antibodies to the RBD of the SARS-CoV-2 S1 subunit have been shown to inhibit interaction with the ACE-2 receptor, confirming S1 subunit especially the RBD as an attractive target for vaccinations or antiviral therapy (13). There is also promising work showing that the RBD may be used to detect presence of neutralizing antibodies present in a patient's bloodstream, consistent with developed immunity after exposure to the SARS-CoV-2 virus (14). Lastly, it has been demonstrated the S Protein can invade host cells through the CD147/EMMPRIN receptor and mediate membrane fusion (15, 16).

Item	Value
References	Wu, F. et al. (2020) Nature 579:265. Tortorici, M.A. and D. Veesler (2019) Adv. Virus Res. 105:93. Bosch, B.J. et al. (2003) J. Virol. 77:8801. Belouzard, S. et al. (2009) Proc. Natl. Acad. Sci. 106:5871. Millet, J.K. and G. R. Whittaker (2015) Virus Res. 202:120. Hodcroft, E.B. et al. (2020) medRxiv https://doi.org/10.1101/2020.10.25.20219063. Bartolini, B. et al. (2020) medRxiv https://doi.org/10.1101/2020.11.28.20237016. Yuan, Y. et al. (2017) Nat. Commun. 8:15092. Walls, A.C. et al. (2010) Cell 180:281. Jiang, S. et al. (2020) Trends. Immunol. https://doi.org/10.1016/j.it.2020.03.007. Ortega, J.T. et al. (2020) EXCLI J. 19:410. Wrapp, D. et al. (2020) Science 367:1260. Tai, W. et al. (2020) Cell. Mol. Immunol. https://doi.org/10.1016/j.it.2020.03.007. Okba, N.M.A. et al. (2020). Emerg. Infect. Dis. https://doi.org/10.3201/eid2607.200841. Wang, X. et al. (2020) https://doi.org/10.1038/s41423-020-0424-9. Wang, K. et al. (2020) ioRxiv https://www.biorxiv.org/content/10.1101/2020.03.14.988345v1.
Long Name	Spike Protein, S1 Subunit
Alternate Names	SARS-CoV-2, Spike S1 Subunit

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