SARS Spike (408-470, 540-573)

What is the structural composition of SARS Spike (408-470, 540-573 aa)?

SARS Spike (408-470, 540-573 aa) comprises two discontinuous segments of the SARS coronavirus spike glycoprotein. This recombinant construct typically includes a C-terminal His-tag and is produced in E. coli expression systems . The 408-470 and 540-573 regions are functionally significant segments of the spike protein. When produced as a recombinant protein, it demonstrates >90% purity when analyzed by SDS-PAGE . These regions are important because they contain elements related to receptor binding and are targeted by neutralizing antibodies.

The protein is typically formulated in PBS without preservatives and should be stored at temperatures between -20°C and -70°C to maintain stability . Researchers should avoid freeze/thaw cycles to prevent protein degradation and loss of activity.

How does SARS Spike (408-470, 540-573) differ from complete spike protein?

The SARS Spike (408-470, 540-573) construct represents specific discontinuous segments of the full-length spike protein, focusing on regions with particular functional or immunological significance. The complete spike protein is considerably larger (approximately 1,255 amino acids) and contains multiple domains, including the N-terminal domain (NTD), receptor-binding domain (RBD), and S2 subunit responsible for membrane fusion .

These selected regions (408-470, 540-573) likely contain important epitopes that are involved in receptor binding and antibody recognition. Working with these specific segments allows researchers to:

• Focus on functionally critical regions without the complexity of the full protein

• Reduce experimental variables associated with the highly glycosylated nature of the complete spike

• Enable more straightforward expression in bacterial systems compared to the full-length protein

• Facilitate specific antibody development against these regions

What purification methods are recommended for SARS Spike (408-470, 540-573)?

For SARS Spike (408-470, 540-573) with a His-tag, affinity chromatography using nickel or cobalt resins is the primary purification method. Based on standard protocols for His-tagged proteins and information from commercial preparations, the following methodology is recommended:

• Primary Purification: Immobilized metal affinity chromatography (IMAC) using Ni-NTA or Co-NTA resins

• Secondary Purification: Size exclusion chromatography to remove aggregates and ensure homogeneity

• Quality Control: SDS-PAGE analysis to confirm >90% purity as typically reported for commercial preparations

Buffer compositions typically involve PBS (pH 7.4) for final formulation, without preservatives that might interfere with downstream applications . For specific research applications requiring higher purity, additional chromatography steps such as ion exchange may be incorporated.

How do mutations in the 408-470 and 540-573 regions affect antibody neutralization?

Mutations in these regions can significantly impact antibody neutralization efficacy. Recent structural and functional studies have mapped the epitope landscape of neutralizing antibodies (nAbs) on the spike protein, revealing how specific mutations can enable immune evasion .

The receptor-binding domain (RBD) of the spike protein, which partially overlaps with the 408-470 region, is a prime target for neutralizing antibodies. Mutations in these regions can:

• Alter antibody binding affinity

• Completely abrogate antibody recognition

• Maintain viral fitness while escaping immune recognition

Research has shown that certain mutations create structural and antigenic variations that reduce antibody neutralization potency . This understanding is crucial for developing escape-resistant antibody therapeutics and vaccines.

As noted in recent studies: "The potency of therapeutic antibodies and vaccines partly depends on how readily the virus can escape neutralization. Recent structural and functional studies have mapped the epitope landscape of nAbs on the spike protein, which illustrates the footprints of several nAbs and the site of escape mutations" .

What mass spectrometry approaches are effective for detecting SARS Spike (408-470, 540-573) in biological samples?

Untargeted nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS/MS) has proven effective for detecting SARS-CoV-2 spike protein fragments in biological samples. Based on recent methodological studies, the following approach is recommended:

• Sample Preparation:

  • Virus inactivation

  • Optional deglycosylation (depending on research question)

  • Protein digestion (typically trypsin)

• MS Analysis Parameters:

  • Nano-flow liquid chromatography coupled to ESI-MS/MS

  • Multiple reaction monitoring (MRM) for targeted detection

  • Data-dependent acquisition for discovery-based approaches

• Key Peptides for Detection:
  From the spike protein region, specific peptides have demonstrated good detection sensitivity, including:

  • VGGNYNYLYR

  • QIAPGQTGK

  • FLPFQQFGR

  • PFAMQMAYR

The limit of detection (LOD) varies by peptide, with some detectable at concentrations as low as 10-100 pg of purified protein . This sensitivity makes the approach viable for clinical sample analysis, though sensitivity may be reduced in complex biological matrices.

How does glycan engineering of these regions influence SARS spike immunogenicity?

Glycan engineering of SARS spike regions, including those containing 408-470 and 540-573 segments, significantly impacts immunogenicity and can be strategically employed to direct immune responses toward specific conserved epitopes.

Recent studies have shown that introducing N-linked glycans onto receptor-binding motif (RBM) surfaces of the SARS-CoV-2 RBD can:

• Redirect immune responses toward the more conserved core-RBD regions

• Elicit higher proportions of core-RBD-specific germinal center B cells

• Generate broadly neutralizing antibody responses against multiple SARS-like coronaviruses

A notable finding is that glycan-modified RBD constructs demonstrated significant neutralizing activity not only against SARS-CoV-2 but also SARS-CoV and bat WIV1-CoV . This approach represents a promising strategy for developing pan-sarbecovirus vaccines.

The research indicates: "These results have implications for the design of SARS-like virus vaccines" , suggesting that strategic glycan modification of spike regions can potentially produce broader protection against emerging coronaviruses.

What are the comparative differences between corresponding regions in SARS-CoV and SARS-CoV-2 spike proteins?

The regions corresponding to 408-470 and 540-573 in SARS-CoV and SARS-CoV-2 exhibit both structural conservation and notable variations that affect antibody recognition and receptor binding.

Key differences include:

These differences are particularly relevant for developing broadly neutralizing antibodies and pan-coronavirus vaccines. Understanding the structural conservation between these viruses is essential for designing immunogens that can elicit broadly protective responses .

What experimental approaches are recommended for studying antibody binding to SARS Spike (408-470, 540-573)?

Several complementary techniques are recommended for comprehensive characterization of antibody binding to SARS Spike (408-470, 540-573):

• Surface Plasmon Resonance (SPR)

  • Provides real-time binding kinetics (ka, kd) and affinity (KD)

  • Allows comparison of multiple antibodies against the same antigen

  • Enables epitope competition studies

• ELISA-Based Methods

  • Quantitative assessment of binding

  • High-throughput screening capability

  • Can be adapted for epitope mapping through peptide arrays

• Structural Analysis

  • X-ray crystallography of antibody-antigen complexes

  • Cryo-electron microscopy for visualization of binding conformations

  • Hydrogen-deuterium exchange mass spectrometry for epitope mapping

• Functional Neutralization Assays

  • Pseudovirus neutralization assays

  • Cell-cell fusion inhibition assays

  • ACE2-spike protein interaction inhibition assays

These approaches should be used in combination to provide complementary data on binding specificity, affinity, and functional consequences of antibody binding to these regions.

How can SARS Spike (408-470, 540-573) be utilized in diagnostic assay development?

SARS Spike (408-470, 540-573) can be strategically employed in several diagnostic formats:

• Antibody Detection Assays

  • ELISA-based detection of IgM and IgG antibodies targeting these regions

  • Lateral flow immunoassays for rapid testing with reported sensitivity of 88.66% and specificity of 90.63% for spike-based detection

  • Potential differentiation between recent (IgM, 5-10 days post-infection) and past (IgG, 14-21 days) infections

• Advanced Biosensing Platforms

  • Graphene-based field-effect transistor (FET) biosensors coated with antibodies against spike protein regions demonstrate ultra-high sensitivity (1 fg/ml)

  • These platforms enable rapid, highly sensitive detection from clinical specimens

• Multiplex Assays

  • Incorporation of multiple spike protein regions for improved sensitivity and specificity

  • Differentiation between SARS-CoV and SARS-CoV-2 antibodies based on region-specific recognition

When developing diagnostic assays using these regions, researchers should consider:

• The temporal dynamics of antibody responses

• Cross-reactivity with other coronaviruses

• Validation against gold standard methods such as RT-PCR

• Impact of viral variants on test performance

What quality control measures should be implemented when working with SARS Spike (408-470, 540-573)?

Comprehensive quality control is essential when working with SARS Spike (408-470, 540-573) to ensure experimental reproducibility and reliability:

• Purity Assessment

  • SDS-PAGE analysis (target >90% purity)

  • Mass spectrometry to confirm molecular weight and sequence integrity

  • Endotoxin testing for preparations intended for immunological studies

• Functional Validation

  • ELISA-based binding assays with known antibodies

  • ACE2 binding assays (if applicable to the region)

  • Circular dichroism for secondary structure confirmation

• Storage Stability Monitoring

  • Avoid freeze/thaw cycles

  • Store at -20°C to -70°C in working aliquots

  • Perform periodic quality checks on stored material

• Batch Consistency

  • Implement reference standards for batch-to-batch comparison

  • Document lot-specific validation data

  • Consider activity normalization between batches for critical applications

These measures help ensure that experimental results are attributable to the biological properties of the protein rather than quality variations or degradation products.

What are the considerations for designing antibody escape studies using SARS Spike (408-470, 540-573)?

When designing antibody escape studies focusing on SARS Spike (408-470, 540-573), researchers should consider several methodological aspects:

• Selection of Antibody Panels

  • Include antibodies targeting diverse epitopes within the regions

  • Consider both monoclonal and polyclonal antibodies

  • Include antibodies with known neutralizing capacity

• Mutation Analysis Approaches

  • Deep mutational scanning to systematically identify escape mutations

  • Structural analysis to predict potential escape mutations

  • Focus on naturally occurring variants in these regions

• Experimental Design Considerations

  • Use both binding assays (ELISA, SPR) and functional assays (neutralization)

  • Implement controls to distinguish affinity changes from complete escape

  • Consider competitive binding assays to map epitope relationships

• Data Analysis and Interpretation

  • Create comprehensive escape maps for each antibody tested

  • Correlate escape mutations with structural data on antibody-antigen complexes

  • Analyze the fitness consequences of escape mutations

As noted in recent research: "These escape maps are a valuable tool to predict SARS-CoV-2 fitness, and in conjunction with the structures of the spike-nAb complex, they can be utilized to facilitate the rational design of escape-resistant antibody therapeutics and vaccines" .

How can SARS Spike (408-470, 540-573) contribute to pan-coronavirus vaccine development?

The SARS Spike (408-470, 540-573) regions contain epitopes that may be valuable for developing broadly protective vaccines against multiple coronaviruses. Current research indicates several promising approaches:

• Glycan Shielding Strategies

  • Strategic placement of N-linked glycans on variable regions to direct immune responses toward conserved epitopes

  • Studies show that glycan-modified constructs can elicit antibodies with neutralizing activity against multiple SARS-like coronaviruses

• Epitope-Focused Design

  • Isolating and presenting conserved epitopes from these regions

  • Engineering for improved stability and immunogenicity

  • Multimerization to enhance B-cell responses

• Immunological Considerations

  • Targeting germinal center B cell responses specific to conserved regions

  • Evaluating T-cell epitopes within these regions for cellular immunity

  • Assessment of immune imprinting and original antigenic sin

Research has demonstrated that strategic modifications to spike regions can "elicit higher proportions of the core-RBD-specific germinal center (GC) B cells and antibody responses, thereby manifesting significant neutralizing activity for SARS-CoV, SARS-CoV-2, and the bat WIV1-CoV" .

What are the implications of emerging SARS-CoV-2 variants for research using SARS Spike (408-470, 540-573)?

Emerging SARS-CoV-2 variants pose significant challenges and opportunities for research using SARS Spike (408-470, 540-573):

• Epitope Conservation Analysis

  • Assessment of epitope conservation across variants

  • Identification of invariant regions suitable for broad-spectrum diagnostics

  • Mapping of escape mutations in these specific regions

• Diagnostic Implications

  • Evaluation of diagnostic sensitivity for variant detection

  • Development of variant-specific detection methods

  • Assessment of false-negative rates due to mutations

• Therapeutic Antibody Development

  • Identification of conserved epitopes within these regions

  • Design of antibody cocktails targeting multiple epitopes

  • Structure-guided optimization of antibody binding to accommodate variants

As noted in recent studies: "Mutation in the Spike gene, such as 69‐70del that is amplified by RT-PCR has shown to affect specificity and sensitivity of the assay" . This highlights the importance of continuous monitoring and adaptation of diagnostic and therapeutic approaches based on emerging variants.

What expression systems are optimal for producing SARS Spike (408-470, 540-573) for different applications?

The choice of expression system for SARS Spike (408-470, 540-573) depends on the specific research application:

• E. coli Expression

  • Advantages: High yield, cost-effective, simpler purification

  • Optimal for: Basic binding studies, antibody production, structural analysis

  • Limitations: Lacks eukaryotic post-translational modifications

  • Current use: Commercial preparations typically use E. coli systems

• Mammalian Cell Expression

  • Advantages: Proper folding, glycosylation patterns

  • Optimal for: Functional studies requiring native conformation

  • Limitations: Lower yield, higher cost

  • Recommended cell lines: HEK293, CHO cells

• Insect Cell Expression

  • Advantages: Higher yield than mammalian cells, some post-translational modifications

  • Optimal for: Structural studies requiring glycosylation

  • Systems: Baculovirus expression vector system (BEVS)

• Cell-Free Systems

  • Advantages: Rapid production, avoids cellular toxicity

  • Optimal for: Quick screening of variants

  • Limitations: Lower yield, higher cost

For applications requiring native glycosylation patterns, mammalian expression systems are recommended despite their lower yield, as glycosylation can significantly impact antibody recognition and functional properties.

What analytical techniques best characterize the structural integrity of SARS Spike (408-470, 540-573)?

Multiple complementary analytical techniques should be employed to comprehensively assess the structural integrity of SARS Spike (408-470, 540-573):

For routine quality control, a combination of SDS-PAGE, SEC, and mass spectrometry is typically sufficient. For detailed structural characterization, additional techniques such as CD and DSC provide valuable information about folding and stability.

Mass spectrometry approaches, as described in recent research, can detect specific peptides from the spike protein with high sensitivity, making it valuable for both structural characterization and detection in biological samples .