Recombinant SARS-CoV-2 P.1 Spike RBD His-tag Protein, CF

Brazilian Variant. K417T, E484K, N501Y
Catalog # Availability Size / Price Qty
10775-CV-100
Recombinant SARS-CoV-2 P.1 Spike RBD His-tag Protein Binding Activity.
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Recombinant SARS-CoV-2 P.1 Spike RBD His-tag Protein, CF Summary

Product Specifications

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.
Activity
Measured by its binding ability in a functional ELISA with Recombinant Human ACE-2 His-tag (Catalog # 933-ZN).
Source
Human embryonic kidney cell, HEK293-derived sars-cov-2 Spike RBD protein
Arg319-Phe541(Lys417Thr, Glu484Lys, Asn501Tyr), with a C-terminal 6-His tag
Accession #
N-terminal Sequence
Analysis
Protein identity confirmed by mass spectrometry.
Predicted Molecular Mass
26 kDa
SDS-PAGE
32-40 kDa, under reducing conditions

Product Datasheets

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.

10775-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.

Data Images

Binding Activity Recombinant SARS-CoV-2 P.1 Spike RBD His-tag Protein Binding Activity. View Larger

Recombinant SARS-CoV-2 P.1 Spike RBD His-tag (Catalog # 10775-CV) binds Recombinant Human ACE-2 His-tag (933-ZN) in a functional ELISA.

SDS-PAGE Recombinant SARS-CoV-2 P.1 Spike RBD His-tag Protein SDS PAGE. View Larger

2 μg/lane of Recombinant SARS-CoV-2 P.1 Spike RBD His-tag (Catalog # 10775-CV) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 32-40 kDa.

Surface Plasmon Resonance (SPR) Binding of ACE-2 to Brazilian variant P.1 Spike RBD protein by surface plasmon resonance (SPR). View Larger

Recombinant SARS-CoV-2 Brazilian variant P.1 Spike RBD 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 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= 1.625 nM. (Biacore T200).

Reconstitution Calculator

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 RBD

SARS-CoV-2, which causes the global pandemic coronavirus disease 2019 (Covid-19), belongs to a family of viruses known as coronaviruses that also include MERS‑CoV and SARS-CoV-1. Coronaviruses are commonly comprised of four structural proteins: Spike protein (S), Envelope protein (E), Membrane protein (M) and Nucleocapsid protein (N) (1). The SARS-CoV-2 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 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). A receptor binding domain (RBD) in the C-terminus of the S1 subunit has been identified and the RBD of SARS-CoV-2 shares 73% amino acid (aa) identity with the RBD of the SARS-CoV-1, but only 22% aa identity with the RBD of MERS‑CoV (6, 7). The low aa sequence homology is consistent with the finding that SARS and MERS‑CoV bind different cellular receptors (8). The RBD of SARS-CoV-2 binds a metallopeptidase, angiotensin-converting enzyme 2 (ACE-2), similar to SARS-CoV-1, but with much higher affinity and faster binding kinetics (9). Before binding to the ACE-2 receptor, structural analysis of the S1 trimer shows that only one of the three RBD domains 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 (10). Polyclonal antibodies to the RBD of the SARS-CoV-2 protein have been shown to inhibit interaction with the ACE-2 receptor, confirming RBD as an attractive target for vaccinations or antiviral therapy (11). 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 (12). Several emerging SARS-CoV-2 genomes have been identified including the P.1 (Brazilian) variant (13). The P.1 variant contains 3 mutations of interest in the RBD domain: K417T, E484K, and N501Y (13). The E484K substitution alone has been shown to confer resistance to several monoclonal antibodies and is responsible for the first confirmed SARS-CoV-2 reinfection (14). Structural analysis points to E484K as a potentially crucial mutation as it creates a new site for hACE-2 binding and may enhance binding affinity (15).

References
  1. Wu, F. et al. (2020) Nature 579:265.
  2. Tortorici, M.A. and D. Veesler (2019) Adv. Virus Res. 105:93.
  3. Bosch, B.J. et al. (2003). J. Virol. 77:8801.
  4. Belouzard, S. et al. (2009) Proc. Natl. Acad. Sci. 106:5871.
  5. Millet, J.K. and G.R. Whittaker (2015) Virus Res. 202:120.
  6. Li, W. et al. (2003) Nature 426:450.
  7. Wong, S.K. et al. (2004) J. Biol. Chem. 279:3197.
  8. Jiang, S. et al. (2020) Trends. Immunol. https://doi.org/10.1016/j.it.2020.03.007.
  9. Ortega, J.T. et al. (2020) EXCLI J. 19:410.
  10. Wrapp, D. et al. (2020) Science 367:1260.
  11. Tai, W. et al. (2020) Cell. Mol. Immunol. https://doi.org/10.1016/j.it.2020.03.007.1.
  12. Okba, N.M.A. et al. (2020) Emerg. Infect. Dis. https://doi.org/10.3201/eid2607.200841.
  13. Ferrareze, P.A.G. et al. (2021) bioRxiv https://doi.org/10.1101/2021.01.27.42689.
  14. Vasques Nonaka, C.K. et al. (2021) Emerg Infect Dis. https://doi.org/10.3201/eid2705.21019.1.
  15. Wang, W.B. et al. (2021) bioRxiv https://doi.org/10.1101/2021.02.17.431566.
Long Name
Spike Receptor Binding Domain
Entrez Gene IDs
3200426 (HCoV-HKU1); 1489668 (SARS-CoV); 43740568 (SARS-CoV-2)
Alternate Names
2019-nCoV Peplomer protein; 2019-nCoV S Protein; 2019-nCoV Spike; COVID-19 Spike; E2; Human coronavirus spike glycoprotein; Peplomer protein; S glycoprotein; S protein; SARS-COV-2 E2; SARS-COV-2 Peplomer protein; SARS-COV-2 S protein; SARS-COV-2 Spike glycoprotein; SARSCOV2 Spike protein; SARS-CoV-2; Severe Acute Respiratory Syndrome Coronavirus 2 Spike Protein; Spike glycoprotein; Spike RBD; Spike S1 RBD; Spike S1; Spike; surface glycoprotein

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