Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of...

Area of Science:

• Virology and Immunology
• Molecular Biology and Biochemistry

Background:

• The emergence of SARS-CoV-2 necessitates tools for antibody characterization.
• Biotin-labeled molecular probes are valuable for isolating and studying antibodies against viral pathogens.

Approach:

• Designed and constructed biotin-labeled probes incorporating SARS-CoV-2 spike protein regions.
• Constructs included purification tags, protease cleavage sites, probe regions (full ectodomain and subregions), and biotin ligase targets.
• Mutants were engineered to abolish ACE2 receptor binding for specific probe applications.

Key Points:

• Achieved high yields of biotinylated probes (up to >5 mg/L for subregions) via transient transfection.
• Characterized probes for antigenicity, ACE2 recognition, and antibody-binding specificities.
• Determined the structure of the spike ectodomain probe using cryo-electron microscopy.

Conclusions:

• Structure-based design combined with efficient purification and biotinylation streamlines probe development.
• Biotinylated SARS-CoV-2 spike probes are effective for antibody characterization and cell sorting.
• This methodology supports the development of diagnostics and therapeutics targeting SARS-CoV-2.