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SARS-CoV-2 Spike RBD Proteins

Spike-RBD-His Unit size Cat. code Docs Qty Price
SARS-CoV2 Spike RBD-His fusion protein
50 µg
his-sars2-rbd
+-
$210.00
Spike-RBD-Fc Unit size Cat. code Docs Qty Price
SARS-CoV2 Spike RBD-Fc fusion protein
50 µg
fc-sars2-rbd
+-
$210.00

SARS-CoV-2 Spike RBD with C-term His or Fc tag

PROTEIN DESCRIPTION

Potential applications of soluble spike proteins
Potential applications of soluble spike proteins

   InvivoGen also offers:

Soluble Human ACE2 protein

The SARS-CoV-2 Spike RBD (receptor binding domain) has been identified as the key viral element allowing the virus docking to the target cells. RDB is recognized by the ACE2 surface membrane receptor [1-3]. RBD is a candidate for subunit prophylactic vaccines against SARS-CoVs [4, 5]. RBD is also at the center of therapeutic approaches, such as the development and testing of small peptide inhibitors or soluble ACE2 to block the SARS-CoV-2 entry into target cells [6].
Spike-RBD-His and Spike-RBD-Fc were generated by fusing the C-terminus of SARS-CoV-2 Spike RBD [R319-F541] to a poly-histidine sequence, and to a human IgG1 Fc region, respectively.
The SARS-CoV-2 viral sequence used is from the Wuhan-Hu-1 (D614) isolate.
These proteins have been produced in CHO cells and purified by affinity chromatography (See Details and Specifications for more information).

APPLICATIONS

  • Vaccination studies: using combinations of Spike protein antigens and adjuvants
  • Antibody screening: finding anti-Spike antibodies that can neutralize the SARS-CoV-2 infection
  • Inhibitor screening: finding small molecules, or antibodies able to block the SARS-CoV-2 RBD interaction with the ACE2 receptor
  • ACE2 cellular expression screening: in primary isolated cells or transfected cells

QUALITY CONTROL

 

Learn more on SARS-CoV-2Learn more about SARS-CoV-2 infection cycle, immune responses, and potential therapeutics.

 

References

1. Li F., 2016. Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3:237-261.
2. Li F. et al., 2005. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 309:1864-1868.
3. Walls A.C. et al., 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 181(2):281-292.e6.
4. Wang N. et al., 2020. Subunit vaccines against emerging pathogenic human coronaviruses. Front. Microbiol. 11:298. DOI: 10.3389/fmicb.2020.00298.
5. Padron-Regalado E., 2020. Vaccines for SARS-CoV-2: Lessons from other coronavirus strains. Infect. Dis. Ther. DOI: 10.1007/s40121-020-00300-x.
6. Monteil V.et al., 2020. Inhibition of SARS-CoV-2 infections in engineered human tissues using clinical-grade soluble human ACE2. Cell. 181:1-9.

Figures

Spike-RBD-His analysis by SDS PAGE
Spike-RBD-His analysis by SDS PAGE

SDS PAGE analysis of the Spike-RBD-His protein. 2 μg of the fusion protein  was loaded on a 12% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad). Detection was performed as per manufacturer’s intructions.

Recognition of Spike-RBD-His by anAnti-SARS-CoV-Spike human IgM
Recognition of Spike-RBD-His by anAnti-SARS-CoV-Spike human IgM

ELISA detection of the SARS-CoV-2 Spike-RBD-His fusion protein with the Anti-SARS-CoV-Spike human IgM. Anti-SARS-CoV-Spike hIgM antibody (5 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of Spike-RBD-His (red curve) or of ACP5-His control protein  (grey  curve) was realized for the capture step. An HRP-labelled anti-His antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

Spike-RBD-Fc analysis by SDS PAGE
Spike-RBD-Fc analysis by SDS PAGE

SDS PAGE analysis of the Spike-RBD-Fc protein.1 μg of the fusion protein was loaded on a 12% Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Bio-Rad). Detection was performed as per manufacturer’s intructions.

Recognition of Spike-RBD-Fc by anAnti-SARS-CoV-Spike human IgM
Recognition of Spike-RBD-Fc by anAnti-SARS-CoV-Spike human IgM

ELISA detection of the SARS-CoV-2 Spike-RBD-Fc fusion protein with the Anti-SARS-CoV-Spike human IgM. Anti-SARS-CoV-Spike hIgM antibody (5 μg/ml) was coated on ELISA plates overnight. A 3-fold serial dilution of Spike-RBD-Fc (red  curve) or of CTLA4-hFc control protein (grey curve) was realized for the capture step. An HRP-labelled anti-hFc antibody (1/1000 dilution) and the HRP substrate OPD (o-phenylenediamine dihydrochloride) were used for the detection step. Absorbance was read at 490 nm.

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Specifications

Spike-RBD-His

  • Protein construction: RBD [R319-F541] from the Spike glycoprotein with a C-terminal poly-histidine tag 
  • Accession sequence: YP_009724390
  • Species: SARS-CoV-2 (2019-nCoV); Wuhan-Hu-1 (D614) isolate
  • Tag: C-terminal poly-histidine (6 x His)
  • Total protein size: 232 a.a.
  • Molecular weight: ~30 kDa (SDS PAGE gel)
  • Purification: Ni2+ affinity chromatography
  • Purity: >95% (SDS PAGE)
  • Quality control:
    - The protein has been validated by ELISA upon incubation with a coated Anti-SARS-CoV-Spike-RBD hIgM mAb (clone CR3022)
    - The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.

Spike-RBD-Fc

  • Protein construction: RBD [R319-F541] from the Spike glycoprotein with a C-terminal human IgG1 Fc tag 
  • Accession sequence: YP_009724390
  • Species: SARS-CoV-2 (2019-nCoV); Wuhan-Hu-1 (D614) isolate
  • Tag: C-terminal human IgG1 Fc
  • Total protein size: 486 a.a.
  • Molecular weight: ~59 kDa (SDS PAGE)
  • Purification: Protein G affinity chromatography
  • Purity: >95% (SDS PAGE)
  • Quality control:
    - The protein has been validated by ELISA upon incubation with a coated Anti-SARS-CoV-Spike-RBD hIgM mAb (clone CR3022)
    - The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cellular assays.
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Contents

Spike-RBD-His and Spike-RBD-Fc contents:

  • 50 μg of lyophilized protein
  • 1.5 ml of endotoxin-free water

room temperature The product is shipped at room temperature.

store Lyophilized protein should be stored at -20 ̊C.

stability Resuspended protein is stable up to 1 month when stored at 4°C, and 1 year when stored at -20°C

Avoid repeated freeze-thaw cycles.

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Details

Spike (S) is a multifunctional glycoprotein that mediates the SARS-CoV-2 entry into the target cells [1, 2]. Most of the current knowledge about the SARS-CoV-2 Spike is based on analogies with findings on the SARS-CoV Spike. At the surface of the virus, Spike is a clove-shaped trimer. Each Spike protomer contains three segments: a large ectodomain, a transmembrane anchor (TM), and a short intracellular tail (IC) [1-3]. The Spike ectodomain contains three critical elements: the S1 subunitthe RBD (receptor binding domain), and the S2 subunit. Two cleavage sites at the S1 and S2 boundary (S1/S2) and in the S2 domain (S2’) play an essential role in the viral entry into host target cells [3-5].

The Spike trimer exists in two structurally distinct conformations: pre-fusion and post-fusion. In its pre-fusion state, the Spike is a "closed" clove-shape with three S1 heads and a trimeric S2 stalk. The S1 "closed" conformation exerts a physical constraint on the S2 subunit until specific proteases cleave the S1/S2 and S2' sites [3]. The RBDs is located in the S1-CTD region and is buried in the inner S1 head-trimer. The S1 "open" conformation is expected to be necessary for binding to the ACE2 receptor at the surface of host target cells [3, 4]. The opening of the S1-CTD is thus expected to be necessary for binding to ACE2. The exact mechanisms driving the opening of an S1-CTD domain and exposition of RBD so that it can bind the ACE2 receptor are not elucidated yet. It has been proposed that the S protein is cleaved into S1 and S2 subunits by proteases, including furin and the host surface-associated transmembrane protease serine 2 (TMPRSS2) [3-5]. S1 binds to ACE2 through RBD, and S2 is further cleaved and activated by TMPRSS2 [3, 4]. Together these actions result in host-viral membrane fusion and release of the viral RNA genome into the host cell cytoplasm.

The previous SARS-CoV and MERS-CoV outbreaks have prompted the search for protective vaccines. Among the different strategies, protein vaccination using the full Spike or its S1 or RBD fragments, have provided encouraging results in pre-clinical studies [6], and some have reached a clinical-stage (phase I) [7]. One clinical trial testing a prophylactic SARS-CoV-2 vaccine based on the full-length Spike mRNA has been launched in March 2020 [NCT04283461].

 

References

1. Li F., 2016. Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3:237-261.
2. Li F. et al., 2005. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 309:1864-1868.
3. Walls A.C. et al., 2020. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 181(2):281-292.e6.
4. Hoffmann M. et al., 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 181:1-16.
5. Hoffmann M.  et al., 2020. A multibasic cleavage site in the Spike protein of SARS-CoV-2 is essential for infection of human lung cells. Molecular Cell. 78:1-6.
6. Wang N. et al., 2020. Subunit vaccines against emerging pathogenic human coronaviruses. Front. Microbiol. 11:298. DOI: 10.3389/fmicb.2020.00298.
7. Padron-Regalado E., 2020. Vaccines for SARS-CoV-2: Lessons from other coronavirus strains. Infect. Dis. Ther. DOI: 10.1007/s40121-020-00300-x.

 

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