CoV-2-S1 (319-541), Fc
Description
Definition and Molecular Architecture
CoV-2-S1 (319-541), Fc is a recombinant protein construct derived from the SARS-CoV-2 spike glycoprotein. It consists of two key components:
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Receptor-binding domain (RBD): The S1 subunit (amino acids 319–541) responsible for binding to the human angiotensin-converting enzyme 2 (ACE2) receptor .
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Fc tag: The crystallizable fragment (Fc) of human IgG1 fused to the C-terminus to enhance protein stability, solubility, and detection in experimental assays .
This engineered protein is produced in HEK293 cells and purified using affinity chromatography, achieving >95% purity .
Key Domains and Interactions
Structural Comparison with Full-Length Spike Protein
The RBD (319–541) resides within the S1 subunit’s C-terminal domain (CTD), distinct from the N-terminal domain (NTD, residues 14–305), which harbors a galectin-like fold implicated in monocyte activation . Unlike the full S1 subunit, the isolated RBD lacks immune-activating properties but retains ACE2 affinity .
Table 1: Experimental Uses
Table 2: Biophysical Properties
| Property | Specification |
|---|---|
| Molecular Weight | ~60 kDa (SDS-PAGE) |
| Binding Affinity | High-affinity interaction with ACE2 ( in nM range) . |
| Purity | ≥95% (verified by SDS-PAGE and SEC) . |
Immune Activation Profile
While the full S1 subunit activates monocytes/macrophages to secrete cytokines like IL-6 and TNF-α , the isolated RBD (319–541) does not trigger immune responses directly . This specificity makes it ideal for ACE2-binding studies without confounding inflammatory effects.
Critical Residues for ACE2 Binding
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E484: Stabilizes ionic bonds with ACE2 K31 .
Mutations in these residues (e.g., E484K in variants of concern) alter binding kinetics and immune evasion .
Clinical Implications
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Vaccine Design: The RBD’s high conservation (100% identity across SARS-CoV-2 variants) supports its use in pan-coronavirus vaccines .
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Antiviral Therapeutics: Peptides targeting the RBD-ACE2 interface show promise in blocking viral entry .
Limitations and Future Directions
Product Specs
The novel coronavirus responsible for the COVID-19 pandemic, initially named 2019-nCoV, was first identified in Wuhan, China, in December 2019. It likely originated from a seafood market. This virus is closely related to a bat coronavirus (SARS-CoV-2) discovered in 2018, sharing 87% of its genetic sequence. Both viruses utilize the ACE2 receptor (angiotensin-converting enzyme 2) in humans for cellular entry, though variations exist. While bats are considered the likely natural reservoir, an intermediary animal host, potentially from the seafood market, is suspected. Research indicates that the virus's spike glycoprotein may be a result of recombination between a bat coronavirus and another unknown coronavirus.
This product consists of the Receptor Binding Domain (RBD) of the SARS-CoV-2 Spike Glycoprotein S1, derived from the Wuhan-Hu-1 strain (amino acids 319-541). It is recombinantly produced in HEK293 cells and features a C-terminal Fc tag for purification and detection purposes.
The product is provided as a lyophilized powder, which means it has been freeze-dried to remove moisture for stability.
The lyophilized CoV-S1 RBD protein is formulated in a solution of 1x PBS (phosphate-buffered saline) at a pH of 7.4, with 5% trehalose added as a stabilizing agent.
The lyophilized CoV-2 S1 Glycoprotein RBD is stable at room temperature for up to three weeks. However, for long-term storage, it is recommended to store it desiccated (dry) at a temperature below -18°C. After reconstitution (adding liquid to the powder), the protein can be stored at 4°C for 2-7 days. For extended storage after reconstitution, store at -18°C. To ensure optimal stability during long-term storage, consider adding a carrier protein such as HSA or BSA at a concentration of 0.1%. Avoid repeated freeze-thaw cycles to maintain protein integrity.
The purity of the protein is greater than 95%, as determined by SDS-PAGE analysis, a widely used technique for separating and analyzing protein purity based on size.
HEK293 Cells.
Purified by Protein-G chromatographic technique.
Q&A
Basic Research Questions
What structural and functional features define CoV-2-S1 (319-541), Fc?
The CoV-2-S1 (319-541), Fc construct comprises the receptor-binding domain (RBD) of SARS-CoV-2’s spike protein (residues 319–541) fused to an Fc domain for enhanced stability and detection in assays. Key features include:
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ACE2-binding capability: The RBD mediates viral entry by binding ACE2 .
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Fc domain utility: Enhances protein dimerization, extends serum half-life, and facilitates detection via anti-Fc antibodies in assays .
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Critical epitopes: Residues L455 and F456 in this region are linked to immune escape and ACE2 binding affinity .
How should researchers design experiments to assess S1-induced immune cell activation?
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Immobilization: Coat polystyrene wells with recombinant S1-Fc (10 µg/mL) to mimic cell-surface presentation, as passive adsorption preserves conformational epitopes .
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Controls: Include S2, S1/S2 trimer, and Fc-only proteins to isolate S1-specific effects .
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Cell types: Prioritize monocytes (over pDC/mDC) due to their dominant role in cytokine release syndrome (CRS) .
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Cytokine profiling: Use multiplex assays targeting IL-6, TNF-α, CXCL10, and CCL3/4, which are consistently elevated in S1-activated monocytes .
Why do some studies report conflicting cytokine profiles for S1 subunits?
Discrepancies arise from:
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Protein constructs: Fc-tagged vs. His-tagged S1 (e.g., R&D Systems #10500-Cv vs. NativeAntigen Co. REC31806) may differ in folding or glycosylation .
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Endotoxin levels: Use only endotoxin-free preparations (<0.1 EU/µg) to avoid TLR4-driven artifacts .
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IL-3 priming: IL-3 augments IL-6 secretion but suppresses baseline TNF-α/IL-1β in monocytes .
Advanced Research Questions
How can researchers resolve contradictions in S1’s role in ACE2 binding vs. innate immune activation?
The S1 subunit has dual roles:
| Function | Domain Responsible | Key Residues/Features |
|---|---|---|
| ACE2 binding | CTD/RBD (residues 319–541) | L455, F456, ACE2 interface |
| Innate immune activation | NTD (residues 1–318) | Galectin-3-like fold |
Methodological approach:
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Use truncation mutants (e.g., NTD-only or RBD-only) to decouple these functions .
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Employ competitive inhibition with galectin-3 blockers to confirm NTD’s role in monocyte activation .
What strategies optimize recombinant S1-Fc for mechanistic studies?
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Construct design: Use HEK293-derived proteins for human-like glycosylation .
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Multimerization: Pair Fc-tagged RBD with PP7 bacteriophage VLPs to enhance immune cell engagement .
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Validation: Verify ACE2 binding via SPR/BLI and innate activation via monocyte IL-6 assays .
How do L455F/F456L (“FLip”) mutations in S1 impact antibody evasion and ACE2 affinity?
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ACE2 binding: FLip mutations synergistically increase binding affinity by 2.3-fold (Kd reduction from 14.2 nM to 6.1 nM) .
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Antibody evasion: Disrupts IGHV3-53/3-66 antibody binding by altering RBD’s epitope topography .
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Experimental validation: Use pseudovirus neutralization assays with convalescent plasma to quantify escape .
Data Contradiction Analysis
Why does S1 fail to induce IL-8 or VEGF in some models despite their association with COVID-19?
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Cell-type specificity: S1 activates monocytes but not endothelial cells (primary sources of IL-8/VEGF) .
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Signaling context: IL-8/VEGF require NF-κB/STAT3 co-activation, which S1 alone may not trigger .
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Solution: Combine S1 with endothelial co-cultures or add TLR agonists (e.g., LPS) to model in vivo complexity .
How to address variability in S1-induced CCL2/MCP-1 responses across studies?
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Threshold effects: CCL2 induction by S1 is dose-dependent (detectable ≥5 µg/mL) .
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Donor heterogeneity: Monocyte CD14+/CD16+ subsets exhibit differential chemokine responses .
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Standardization: Pre-screen donors for ACE2 expression and normalize protein batches via ELISA .
Methodological Recommendations
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Cytokine assay: Use Luminex/HCBA platforms for simultaneous quantification of 15+ cytokines (Fig 1/2, ).
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Negative controls: Include S2 subunit (residues 686–1211) to confirm S1-specific effects .
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Structural analysis: Perform cryo-EM to map S1-Fc’s galectin-3-like fold and antibody escape mutations .
Product Science Overview
The Coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to significant global health challenges. One of the critical components of SARS-CoV-2 is the spike (S) glycoprotein, which plays a pivotal role in the virus’s ability to infect host cells. This article delves into the background of the Spike Glycoprotein-S1 Receptor Binding Domain (RBD) (319-541 a.a), Fc Recombinant, a crucial element in understanding and combating COVID-19.
The spike glycoprotein of SARS-CoV-2 is a type I membrane protein that facilitates the virus’s entry into host cells. It is composed of two subunits: S1 and S2. The S1 subunit contains the receptor-binding domain (RBD), which is responsible for binding to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells . The specific region of interest, 319-541 amino acids (a.a), is within the S1 subunit and is critical for the virus’s attachment and entry into cells .
The RBD within the S1 subunit is a major target for neutralizing antibodies. These antibodies can block the interaction between the spike protein and the ACE2 receptor, thereby preventing the virus from entering host cells . This makes the RBD a focal point for vaccine development and therapeutic interventions.
The Fc recombinant fusion involves attaching the Fc region of an antibody to the RBD. This fusion enhances the stability and half-life of the RBD, making it more effective for use in vaccines and therapeutic applications . The Fc region also facilitates the recruitment of immune cells, enhancing the overall immune response against the virus.
- Vaccine Development: The RBD-Fc fusion protein is used in vaccine formulations to elicit a strong immune response. By presenting the RBD in a stable and immunogenic form, these vaccines can effectively stimulate the production of neutralizing antibodies .
- Therapeutic Antibodies: Monoclonal antibodies targeting the RBD have shown efficacy in neutralizing the virus and are used in treating COVID-19 patients .
- Diagnostic Tools: The RBD-Fc fusion protein is also employed in diagnostic assays to detect antibodies against SARS-CoV-2 in patient samples .
