Recombinant SARS-CoV-2 B.1.1.7 S RBD Avi-tag Protein, CF

Biotinylated Recombinant SARS-CoV-2 B.1.1.7 N501Y Spike RBD His-tag Avi-tag (Catalog # AVI10730) binds Recombinant Human ACE-2 Fc Chimera (10544-ZN) in a functional ELISA.

2 μg/lane of Biotinylated Recombinant SARS-CoV-2 B.1.1.7 S RBD Avi-tag Protein (Catalog # AVI10730) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing bands at 34-38 kDa.

• Reactivity: V
• Applications: Bioactivity
• Format: Carrier-Free

Recombinant SARS-CoV-2 B.1.1.7 S RBD Avi-tag Protein, CF Summary

• Additional Information: His-tag Biotinylated N501Y Mutation found in Alpha, Beta, and Gamma Variants
• Details of Functionality: Measured by its binding ability in a functional ELISA with Recombinant Human ACE-2 Fc Chimera (Catalog # 10544-ZN). The biotin to protein ratio is greater than 0.7 as determined by the HABA assay.
• Source: Human embryonic kidney cell, HEK293-derived sars-cov-2 Spike RBD protein
  

  SARS-CoV-2 B.1.1.7 Spike RBD (Arg319-Phe541) (Asn501Tyr) Accession # YP_009724390.1	6-His tag	Avi-tag
  N-terminus		C-terminus

• Accession #: YP_009724390.1
• N-terminal Sequence: Arg319
• Structure / Form: Biotinylated via Avi-tag
• Protein/Peptide Type: Recombinant Proteins
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <0.10 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Bioactivity
  • Bioactivity2
• Theoretical MW: 29 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 36-40 kDa, under reducing conditions.

Packaging, Storage & Formulations

• 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.
• Buffer: Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Reconstitution Instructions: Reconstitute at 500 μg/mL in PBS.

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant SARS-CoV-2 B.1.1.7 S RBD Avi-tag Protein, CF

• Spike RBD

Background

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 B 1.1.7 (United Kingdom) variant (13). The B 1.1.7 variant contains a significant mutation of interest in the RBD domain, N501Y, which has been shown to result in enhanced binding affinity for hACE‑2 (14). Additionally, the B 1.1.7 variant appears to more easily transmissible, exhibit increased viral loads and, potentially, be associated with higher mortality rates compared to preexisting variants (13, 15). Our Avi-tag Biotinylated SARS-CoV-2 Spike B.1.1.7 RBD features biotinylation at a single site contained within the Avi-tag, a unique 15 amino acid peptide. Protein orientation will be uniform when bound to streptavidin-coated surface due to the precise control of biotinylation and the rest of the protein is unchanged so there is no interference in the protein's bioactivity.

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. Kidd, M. et al. (2021) J Infect. Dis. https://doi.org/10.1093/infdis/jiab082.
14. Zahradník, J. et al. (2021) bioRxiv https://doi.org/10.1101/2021.01.06.425392.
15. Davies, N.G. (2020) medRxiv doi:10.1101/2020.12.24.20248822.

Bioinformatics

• Uniprot:
  • YP_009724390.1(Rabbit)