SARS Spike (306-515), Sf9

What is SARS Spike (306-515), Sf9 protein?

SARS Spike (306-515), Sf9 is a recombinant protein fragment representing amino acids 306-515 of the SARS coronavirus Spike glycoprotein. It is produced in Sf9 Baculovirus expression system, resulting in a single, glycosylated polypeptide chain containing 219 amino acids with a molecular mass of 24.7kDa. The protein is engineered with a 6-amino acid His-tag at the C-terminus for purification purposes and is isolated using proprietary chromatographic techniques .

How does SARS Spike (306-515) differ from the full-length Spike protein?

The SARS Spike (306-515) represents a specific fragment of the full-length Spike protein, which spans amino acids 14-1195 and has a molecular mass of approximately 131.9kDa . While the full-length protein contains all functional domains including the complete receptor-binding domain (RBD), transmembrane domain, and fusion peptides, the 306-515 fragment contains a critical portion of the RBD that interacts with the human ACE2 receptor. This truncated version allows researchers to focus on receptor interaction studies without the complexity of working with the entire protein structure .

What are the optimal storage conditions for SARS Spike (306-515), Sf9?

For optimal stability and activity retention:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• Store frozen at -20°C for longer periods of time

• For long-term storage, add a carrier protein (0.1% HSA or BSA)

• Avoid multiple freeze-thaw cycles as they may compromise protein integrity

How can SARS Spike (306-515), Sf9 be used in binding assays?

The SARS Spike (306-515) fragment can be effectively employed in binding assays to study virus-host interactions. Researchers typically use the following methodologies:

• ELISA-based binding studies: Coat plates with purified ACE2 receptor and detect binding using the His-tagged Spike fragment followed by anti-His antibody detection. This allows for quantitative assessment of binding affinity and can be used to screen potential inhibitors .

• Surface Plasmon Resonance (SPR): Immobilize either the Spike fragment or ACE2 on a sensor chip and measure real-time binding kinetics, including association (kon) and dissociation (koff) rate constants.

• Flow cytometry: Label the Spike fragment with fluorescent dyes or use antibody detection systems to analyze binding to ACE2-expressing cells and quantify receptor density.

The biological activity of the protein can be verified by measuring its binding ability in functional ELISA with Human ACE2, similar to the verification method used for the full-length Spike protein .

What methods can be used to confirm the purity and integrity of SARS Spike (306-515), Sf9?

To ensure experimental reproducibility, researchers should verify protein quality using multiple approaches:

• SDS-PAGE: Assess protein purity (should be >85% as typically provided by suppliers) and confirm the molecular weight of approximately 24.7kDa .

• Western Blotting: Confirm identity using anti-His tag or specific anti-SARS Spike antibodies.

• Mass Spectrometry: Verify the exact molecular mass and confirm post-translational modifications, particularly glycosylation patterns.

• Circular Dichroism (CD): Evaluate secondary structure integrity and thermal stability.

• Size Exclusion Chromatography: Detect potential aggregation or degradation products before experimental use.

How can SARS Spike (306-515), Sf9 be utilized in cross-reactivity studies with antibodies against different coronavirus strains?

The SARS Spike (306-515) fragment provides an excellent tool for cross-reactivity studies due to its conserved functional domains. Researchers can implement the following methodologies:

• Comparative ELISA panels: Create microarray panels with Spike fragments from different coronaviruses (SARS-CoV-1, SARS-CoV-2, MERS-CoV) to test antibody cross-reactivity and specificity.

• Epitope mapping: Combine with peptide arrays or hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conserved epitopes across coronavirus strains that could be targeted for broad-spectrum therapeutic development.

• Competition assays: Design experiments where antibodies against different coronavirus Spike proteins compete for binding to SARS Spike (306-515), providing insights into shared recognition determinants .

These approaches allow researchers to identify antibodies with potential cross-protective effects against multiple coronavirus strains and help elucidate evolutionary relationships between viral proteins.

What strategies can be employed to study the impact of glycosylation on SARS Spike (306-515), Sf9 functionality?

Glycosylation of the Spike protein plays a crucial role in protein folding, stability, and immune evasion. Researchers can investigate this aspect through:

• Enzymatic deglycosylation: Treat the protein with enzymes like PNGase F or Endo H to remove N-linked glycans, then compare binding affinity and stability with the glycosylated form.

• Expression in different systems: Compare Spike (306-515) expressed in Sf9 cells versus other expression systems (E. coli, mammalian cells) that produce different glycosylation patterns.

• Site-directed mutagenesis: Generate variants with mutations at glycosylation sites to evaluate the contribution of specific glycans to receptor binding and antibody recognition.

• Glycan analysis: Use techniques like mass spectrometry and lectin microarrays to characterize the glycan composition and structure, correlating specific glycoforms with functional properties .

How does SARS Spike (306-515), Sf9 compare to equivalent fragments from SARS-CoV-2 in research applications?

While both SARS-CoV-1 and SARS-CoV-2 Spike proteins bind to the ACE2 receptor, they exhibit important differences in their RBD regions that affect research applications:

Researchers should consider these differences when designing comparative studies or when using SARS-CoV-1 Spike fragments as models for SARS-CoV-2 research .

What methodology should be employed to evaluate how mutations in the Spike RBD affect ACE2 binding?

To systematically analyze how mutations affect receptor interactions, researchers can implement a structured approach:

This multi-parameter assessment provides comprehensive understanding of how specific mutations contribute to altered receptor binding and potential immune escape.

How can SARS Spike (306-515), Sf9 be incorporated into diagnostic development workflows?

The SARS Spike (306-515) fragment can serve as a valuable component in several diagnostic platforms:

• Antibody detection systems: Use as a capture antigen in ELISA or lateral flow immunoassay (LFIA) formats to detect anti-SARS antibodies in patient samples. These approaches have demonstrated high sensitivity (88.66%) and specificity (90.63%) in clinical evaluations .

• Biosensor development: Incorporate into graphene-based field-effect transistor (FET) biosensing platforms, which have shown extraordinary sensitivity (1 fg/ml) when coated with antibodies against SARS Spike protein .

• Calibration standards: Serve as reference material for quantitative assays measuring Spike protein concentrations in research samples.

• Competitive assays: Design diagnostic tests where patient antibodies compete with labeled SARS Spike (306-515) for binding to immobilized ACE2, providing a functional assessment of neutralizing antibody presence .

What methodological considerations are important when using SARS Spike (306-515), Sf9 for antibody development?

When using this protein fragment for generating and characterizing antibodies, researchers should consider:

• Immunization strategies:

  • Use of adjuvants appropriate for glycosylated proteins

  • Prime-boost strategies that may enhance antibody diversity

  • Intrasplenic injection of plasmid DNA encoding the Spike region, which has been reported as an effective approach

• Screening methods:

  • Implement multi-tier screening that first identifies binders then assesses neutralization potential

  • Include counter-screening against related coronavirus Spike proteins to identify cross-reactive antibodies

• Epitope characterization:

  • Perform epitope binning to classify antibodies based on their binding sites

  • Use peptide arrays or hydrogen-deuterium exchange mass spectrometry to precisely map epitopes

• Functional assessments:

  • Evaluate antibody affinity via surface plasmon resonance

  • Test neutralization potency using pseudovirus systems

  • Assess antibody stability and developability characteristics

How can SARS Spike (306-515), Sf9 be used to study the conformational dynamics of receptor binding?

Understanding the dynamic structural changes that occur during receptor binding is crucial for developing intervention strategies. Researchers can utilize several approaches:

• Single-molecule FRET: Label the Spike fragment with donor-acceptor fluorophore pairs at strategic positions to monitor distance changes during ACE2 binding in real-time.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Compare deuterium uptake patterns in free and ACE2-bound states to identify regions undergoing conformational changes.

• Molecular dynamics simulations: Combine experimental data with computational modeling to predict conformational ensembles and energy landscapes.

• Cryo-electron microscopy: Capture different conformational states of the Spike-ACE2 complex under various conditions.

These approaches provide insights into the molecular mechanisms of receptor recognition and can reveal transient conformational states that might be targeted by therapeutics .

What experimental design is recommended for comparing glycosylation patterns between SARS Spike (306-515) expressed in different systems?

Glycosylation can significantly impact protein function and antigenicity. A comprehensive comparative analysis should include:

• Expression in multiple systems:

  • Sf9 insect cells (baculovirus)

  • Mammalian cells (HEK293, CHO)

  • Yeast (Pichia pastoris)

  • Synthesize non-glycosylated version in E. coli as control

• Glycan profiling methodology:

  • Release glycans using PNGase F

  • Label released glycans with fluorescent tags

  • Analyze using hydrophilic interaction liquid chromatography (HILIC) coupled to mass spectrometry

  • Confirm site occupancy using proteomics approaches

• Functional comparison:

  • Measure binding affinity to ACE2 using surface plasmon resonance

  • Evaluate thermal stability using differential scanning fluorimetry

  • Test antibody recognition using a panel of conformational antibodies

• Data analysis framework:

  • Create glycan fingerprints for each expression system

  • Correlate specific glycoforms with functional properties

  • Identify critical glycans that influence receptor binding or antibody recognition