{"doi":"10.1016/j.celrep.2020.108322","title":"Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes","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 ∼0.5 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.","journal":"Cell Reports","year":2020,"id":57847,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":76,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9581,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":229156,"name":"I‐Ting Teng","orcid":"0000-0002-4704-7100","position":1,"is_corresponding":false},{"id":229155,"name":"Adam S. Olia","orcid":"0000-0003-0805-1331","position":2,"is_corresponding":false},{"id":105628,"name":"Gabriele Cerutti","orcid":"0000-0002-9081-7561","position":3,"is_corresponding":false},{"id":105630,"name":"Jason Gorman","orcid":"0000-0002-3775-6560","position":4,"is_corresponding":false},{"id":229154,"name":"Alexandra F. Nazzari","orcid":"0000-0003-1891-8541","position":5,"is_corresponding":false},{"id":105599,"name":"Wei Shi","orcid":"0000-0002-4770-6444","position":6,"is_corresponding":false},{"id":229148,"name":"Yaroslav Tsybovsky","orcid":"0000-0002-2159-5248","position":7,"is_corresponding":false},{"id":105588,"name":"Lingshu Wang","orcid":"0000-0002-8404-4910","position":8,"is_corresponding":false},{"id":229157,"name":"Shuishu Wang","orcid":"0000-0002-0865-4522","position":9,"is_corresponding":false},{"id":229158,"name":"Baoshan Zhang","orcid":"0000-0002-0165-1513","position":10,"is_corresponding":false},{"id":105589,"name":"Yi Zhang","orcid":"0000-0002-8228-2560","position":11,"is_corresponding":false},{"id":229150,"name":"Phinikoula S. Katsamba","orcid":"0000-0003-3981-1604","position":12,"is_corresponding":false},{"id":303498,"name":"Yuliya Petrova","orcid":null,"position":13,"is_corresponding":false},{"id":302416,"name":"Bailey B. Banach","orcid":"0000-0003-0208-5856","position":14,"is_corresponding":false},{"id":302417,"name":"Ahmed S. Fahad","orcid":"0000-0002-5088-9333","position":15,"is_corresponding":false},{"id":105618,"name":"Lihong Liu","orcid":"0000-0003-3128-8513","position":16,"is_corresponding":false},{"id":302418,"name":"Sheila N. López Acevedo","orcid":"0000-0003-2778-4696","position":17,"is_corresponding":false},{"id":302419,"name":"Bharat Madan","orcid":"0000-0002-7376-9991","position":18,"is_corresponding":false},{"id":302420,"name":"Matheus Oliveira de Souza","orcid":"0000-0003-0357-1995","position":19,"is_corresponding":false},{"id":302421,"name":"Xiaoli Pan","orcid":"0000-0002-5033-7272","position":20,"is_corresponding":false},{"id":105619,"name":"Pengfei Wang","orcid":"0000-0003-2454-7652","position":21,"is_corresponding":false},{"id":302422,"name":"Jacy R. Wolfe","orcid":"0000-0001-5206-4135","position":22,"is_corresponding":false},{"id":105635,"name":"Michael T. Yin","orcid":"0000-0002-9346-9056","position":23,"is_corresponding":false},{"id":104013,"name":"David D. Ho","orcid":"0000-0003-1627-149X","position":24,"is_corresponding":false},{"id":105590,"name":"Emily Phung","orcid":"0000-0001-9684-9698","position":25,"is_corresponding":false},{"id":105574,"name":"Anthony DiPiazza","orcid":"0000-0003-1989-8351","position":26,"is_corresponding":false},{"id":105591,"name":"Lauren A. Chang","orcid":"0000-0002-3287-3987","position":27,"is_corresponding":false},{"id":87310,"name":"Olubukola M. Abiona","orcid":"0000-0002-2745-1691","position":28,"is_corresponding":false},{"id":87307,"name":"Kizzmekia S. Corbett","orcid":"0000-0002-2641-4688","position":29,"is_corresponding":false},{"id":108827,"name":"Brandon J. DeKosky","orcid":"0000-0001-6406-0836","position":30,"is_corresponding":false},{"id":87311,"name":"Barney S. Graham","orcid":"0000-0001-8112-0853","position":31,"is_corresponding":false},{"id":105614,"name":"John R. Mascola","orcid":"0000-0002-5293-4695","position":32,"is_corresponding":false},{"id":302423,"name":"John Misasi","orcid":"0000-0001-7169-2639","position":33,"is_corresponding":false},{"id":105610,"name":"Tracy J. Ruckwardt","orcid":"0000-0002-0007-7169","position":34,"is_corresponding":false},{"id":266889,"name":"Nancy J. Sullivan","orcid":"0000-0002-2159-2039","position":35,"is_corresponding":false},{"id":105638,"name":"Lawrence Shapiro","orcid":"0000-0001-9943-8819","position":36,"is_corresponding":false},{"id":105633,"name":"Peter D. Kwong","orcid":"0000-0003-3560-232X","position":37,"is_corresponding":false},{"id":105631,"name":"Tongqing Zhou","orcid":"0000-0002-3935-4637","position":0,"is_corresponding":true}],"reference_count":85,"raw_metadata":null,"created_at":"2026-07-18T21:06:48.611399Z","pmid":"33091382","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}