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Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes

  1. Author:
    Zhou, Tongqing
    Teng, I-Ting
    Olia, Adam S
    Cerutti, Gabriele
    Gorman, Jason
    Nazzari, Alexandra
    Shi, Wei
    Tsybovsky,Yaroslav
    Wang, Lingshu
    Wang, Shuishu
    Zhang, Baoshan
    Zhang, Yi
    Katsamba, Phinikoula S
    Petrova, Yuliya
    Banach, Bailey B
    Fahad, Ahmed S
    Liu, Lihong
    Lopez Acevedo, Sheila N
    Madan, Bharat
    Oliveira de Souza, Matheus
    Pan, Xiaoli
    Wang, Pengfei
    Wolfe, Jacy R
    Yin, Michael
    Ho, David D
    Phung, Emily
    DiPiazza, Anthony
    Chang, Lauren A
    Abiona, Olubukola M
    Corbett, Kizzmekia S
    DeKosky, Brandon J
    Graham, Barney S
    Mascola, John R
    Misasi, John
    Ruckwardt, Tracy
    Sullivan, Nancy J
    Shapiro, Lawrence
    Kwong, Peter D

  2. Author Address

    Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA., Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA., Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA., Bioengineering Graduate Program, The University of Kansas, Lawrence, KS 66045, USA., Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA., Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA., Bioengineering Graduate Program, The University of Kansas, Lawrence, KS 66045, USA; Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA; Department of Chemical Engineering, The University of Kansas, Lawrence, KS 66045, USA., Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address: lss8@columbia.edu., Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA; Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA. Electronic address: pdkwong@nih.gov.,
    1. Year: 2020
    2. Date: Oct 27
    3. Epub Date: 2020 10 12
  2. Journal: Cell reports
    1. 33
    2. 4
    3. Pages: 108322
  4. Type of Article: Article
  5. Article Number: 108322
  6. ISSN: 2211-1247
  1. 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. Published by Elsevier Inc.

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External Sources

  1. View Full Text
  2. DOI: 10.1016/j.celrep.2020.108322
  3. PMID: 33091382
  4. PMCID: PMC7550166
  5. View record in Web of Science®
  6. WOS: 000582721000021
  7. PII : S2211-1247(20)31311-5

Library Notes

  1. Fiscal Year: FY2020-2021
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