What is meant by "Resistant to Furin & Stabilized Prefusion Conformation?" in the description for Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF (Catalog # 10748-CV)?

Furin cleavage resistance is conferred by the Arg682Ser and Arg685Ser mutations. The mutations Lys986Pro and Val987Pro stabilize and promote the prefusion conformation of this Spike protein.

Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF

Name: Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF
Brand: R&D Systems
SKU: 10748-CV

Catalog # 10748-CV

Product Datasheet / COA / SDS

Alpha Variant (UK) Resistant to Furin & Stabilized Prefusion Conformation

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Key Product Details

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

View Full Technical Details for Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF

Product Specifications

Source	Human embryonic kidney cell, HEK293-derived sars-cov-2 Spike protein Val16-Lys1211 (His69 del, Val70 del, Tyr145 del, Asn501Tyr, Ala570Asp, Asp614Gly, Pro681His, Thr716Ile, Ser982Ala, Asp1118His)(Arg682Ser, Arg685Ser, Lys986Pro, Val987Pro), 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	Protein identity is confirmed by mass spectrometry.
Predicted Molecular Mass	134 kDa
SDS-PAGE	148-166 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 B.1.1.7 Spike His-tag Protein, CF Scientific Data Examples

	Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein Binding Activity. Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag (Catalog # 10748-CV) binds Recombinant Human ACE-2 His-tag (933-ZN) in a functional ELISA.
	Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein SDS-PAGE. 2 μg/lane of Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag (Catalog # 10748-CV) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 148-166 kDa.
	Binding of ACE-2 to UK variant B.1.1.7 Spike protein by surface plasmon resonance (SPR). Recombinant SARS-CoV-2 Spike protein UK variant B.1.1.7 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 0.18 nM and 94.3 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=1.185 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.

10748-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

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 glycoprotein that mediates membrane fusion and viral entry. The S protein is homotrimeric, with each ~180-kDa monomer consisting of two subunits, S1 and S2 (2). In SARS-CoV-2, as with most coronaviruses, proteolytic cleavage of the S protein into the 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 (3-5). The S protein of SARS-CoV-2 shares 75% and 29% amino acid (aa) sequence identity with the S protein of SARS-CoV-1 and MERS, respectively.The S Protein of the SARS-CoV-2 virus, like the SARS-CoV-1 counterpart, binds Angiotensin-Converting Enzyme 2 (ACE2), but with much higher affinity and faster binding kinetics through the receptor binding domain (RBD) located in the C-terminal region of S1 (6). Based on structural biology studies, the RBD can be oriented either in the up/standing or down/lying state with the up/standing state associated with higher pathogenicity (7). Polyclonal antibodies to the RBD of the SARS-CoV-2 protein have been shown to inhibit interaction with the ACE2 receptor, confirming RBD as an attractive target for vaccinations or antiviral therapy (8). It has been demonstrated that the S Protein can invade host cells through the CD147/EMMPRIN receptor and mediate membrane fusion (9, 10). A SARS-CoV-2 variant carrying the aa substitution N501Y in the RBD and nine additional mutations in the rest of the spike protein becomes one of the most prevalent mutations found in Covid-19 cases (11-13). This mutant (B.1.1.7 lineage) was originally found in London and the southeast UK but rapidly spread globally (8,9). This new virus variant was reported 56% more transmissible than other preexisting variants (14). The N501Y mutation was also later identified in variants found in South Africa (B.1.351 lineage) and Brazil (P.1 lineage). Although there is no evidence to date that B.1.1.7 causes more severe illness, whether the N501Y mutation in RBD would decrease the efficacy of vaccine-induced immunity is still under investigation.

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. Ortega, J.T. et al. (2020) EXCLI J. 19:410. Yuan, Y. et al. (2017) Nat. Commun. 8:15092. Tai, W. et al. (2020) Cell. Mol. Immunol. https://doi.org/10.1016/j.it.2020.03.007. Wang, X. et al. (2020) https://doi.org/10.1038/s41423-020-0424-9. Wang, K. et al. (2020) bioRxiv https://www.biorxiv.org/content/10.1101/2020.03.14.988345v1. Kozlov, Max (2020) TheScientist https://www.the-scientist.com/news-opinion/new-sars-cov-2-variant-spreading-rapidly-in-uk-68292. Wise, J. (2020) B.M.J. 371:m4857. Tang, J.W. et al. (2020) J. Infect. doi:10.1016/j.jinf.2020.12.024. Davies, N.G. (2020) medRxiv doi:10.1101/2020.12.24.20248822.
Long Name	Spike Protein
Entrez Gene IDs	918758 (HCoV-229E); 2943499 (HCoV-NL63); 39105218 (HCoV-OC43); 37616432 (MERS-CoV); 1489668 (SARS-CoV); 43740568 (SARS-CoV-2)
Alternate Names	2019-nCoV S Protein, 2019-nCoV Spike, COVID-19 Spike, E2, Human coronavirus spike glycoprotein, Peplomer protein, S Protein, S glycoprotein, SARS-COV-2 S protein, SARS-COV-2 Spike glycoprotein, SARS-CoV-2, SARSCOV2 Spike protein, Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein, Spike, Spike glycoprotein, surface glycoprotein

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FAQs

What is meant by "Resistant to Furin & Stabilized Prefusion Conformation?" in the description for Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF (Catalog # 10748-CV)?

Furin cleavage resistance is conferred by the Arg682Ser and Arg685Ser mutations. The mutations Lys986Pro and Val987Pro stabilize and promote the prefusion conformation of this Spike protein.
Recombinant SARS-CoV-2 B.1.1.7 Spike His-tag Protein, CF (Catalog # 10748-CV) states: Alpha Variant (UK) Resistant to Furin & Stabilized Prefusion Conformation. What does Resistant to Furin and Stabilized Prefusion mean?

This spike protein is maintained structurally with stabilizing mutations, K986P and V987P, that promote the prefusion conformation. Additionally, two mutations, R682S and R685S, are included which eliminate a Furin protease cleavage site between the S1 and S2 subunits.