Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes.
Cell Rep
; 33(4): 108322, 2020 10 27.
Article
in English
| MEDLINE | ID: covidwho-888426
Preprint
This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
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This scientific journal article is probably based on a previously available preprint. It has been identified through a machine matching algorithm, human confirmation is still pending.
See preprint
ABSTRACT
Biotin-labeled molecular probes, comprising specific regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. Here, we design constructs incorporating an N-terminal purification tag, a site-specific protease-cleavage site, the probe region of interest, and a C-terminal sequence targeted by biotin ligase. Probe regions include full-length spike ectodomain as well as various subregions, and we also design mutants that eliminate recognition of the angiotensin-converting enzyme 2 (ACE2) receptor. Yields of biotin-labeled probes from transient transfection range from â¼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes are characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe is determined by cryoelectron microscopy. We also characterize antibody-binding specificities and cell-sorting capabilities of the biotinylated probes. Altogether, structure-based design coupled to efficient purification and biotinylation processes can thus enable streamlined development of SARS-CoV-2 spike ectodomain probes.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Pneumonia, Viral
/
Molecular Probes
/
Coronavirus Infections
/
Antibodies, Neutralizing
/
Spike Glycoprotein, Coronavirus
/
Antibodies, Viral
Limits:
Humans
Language:
English
Journal:
Cell Rep
Year:
2020
Document Type:
Article
Affiliation country:
J.celrep.2020.108322
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