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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

View ORCID ProfileTongqing Zhou, I-Ting Teng, Adam S. Olia, Gabriele Cerutti, Jason Gorman, Alexandra Nazzari, Wei Shi, Yaroslav Tsybovsky, Lingshu Wang, Shuishu Wang, Baoshan Zhang, Yi Zhang, Phinikoula S. Katsamba, Yuliya Petrova, Bailey B. Banach, Ahmed S. Fahad, Lihong Liu, Sheila N. Lopez Acevedo, Bharat Madan, Matheus Oliveira de Souza, Xiaoli Pan, Pengfei Wang, Jacy R. Wolfe, Michael Yin, David D. Ho, Emily Phung, Anthony DiPiazza, Lauren Chang, Olubukula Abiona, Kizzmekia S. Corbett, Brandon J. DeKosky, Barney S. Graham, View ORCID ProfileJohn R. Mascola, John Misasi, Tracy Ruckwardt, Nancy J. Sullivan, Lawrence Shapiro, View ORCID ProfilePeter D. Kwong
doi: https://doi.org/10.1101/2020.06.22.166033
Tongqing Zhou
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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  • ORCID record for Tongqing Zhou
I-Ting Teng
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Adam S. Olia
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Gabriele Cerutti
2Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
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Jason Gorman
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Alexandra Nazzari
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Wei Shi
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Yaroslav Tsybovsky
3Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA
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Lingshu Wang
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Shuishu Wang
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Baoshan Zhang
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Yi Zhang
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Phinikoula S. Katsamba
2Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
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Yuliya Petrova
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Bailey B. Banach
4Bioengineering Graduate Program, The University of Kansas, Lawrence, KS 66045, USA
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Ahmed S. Fahad
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Lihong Liu
6Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA
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Sheila N. Lopez Acevedo
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Bharat Madan
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Matheus Oliveira de Souza
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Xiaoli Pan
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Pengfei Wang
6Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA
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Jacy R. Wolfe
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
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Michael Yin
6Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA
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David D. Ho
6Aaron Diamond AIDS Research Center, Columbia University Vagelos College of Physicians and Surgeons, New York, NY 10032, USA
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Emily Phung
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Anthony DiPiazza
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Lauren Chang
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Olubukula Abiona
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Kizzmekia S. Corbett
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Brandon J. DeKosky
4Bioengineering Graduate Program, The University of Kansas, Lawrence, KS 66045, USA
5Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, KS 66045, USA
7Department of Chemical Engineering, The University of Kansas, Lawrence, KS 66045, USA
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Barney S. Graham
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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John R. Mascola
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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John Misasi
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Tracy Ruckwardt
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Nancy J. Sullivan
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Lawrence Shapiro
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
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  • For correspondence: lss8@columbia.edu pdkwong@nih.gov
Peter D. Kwong
1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
2Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA
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  • For correspondence: lss8@columbia.edu pdkwong@nih.gov
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Summary

Biotin-labeled molecular probes, comprising specific regions of the SARS-CoV-2 spike, would be helpful in the isolation and characterization of antibodies targeting this recently emerged pathogen. To develop such probes, we designed 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 included full-length spike ectodomain as well as various subregions, and we also designed mutants to eliminate recognition of the ACE2 receptor. Yields of biotin-labeled probes from transient transfection ranged from ∼0.5 mg/L for the complete ectodomain to >5 mg/L for several subregions. Probes were characterized for antigenicity and ACE2 recognition, and the structure of the spike ectodomain probe was determined by cryo-electron microscopy. We also characterized 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.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵9 Lead Contact: Peter D. Kwong (pdkwong{at}nih.gov)

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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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
Tongqing Zhou, I-Ting Teng, Adam S. Olia, Gabriele Cerutti, Jason Gorman, Alexandra Nazzari, Wei Shi, Yaroslav Tsybovsky, Lingshu Wang, Shuishu Wang, Baoshan Zhang, Yi Zhang, Phinikoula S. Katsamba, Yuliya Petrova, Bailey B. Banach, Ahmed S. Fahad, Lihong Liu, Sheila N. Lopez Acevedo, Bharat Madan, Matheus Oliveira de Souza, Xiaoli Pan, Pengfei Wang, Jacy R. Wolfe, Michael Yin, David D. Ho, Emily Phung, Anthony DiPiazza, Lauren Chang, Olubukula Abiona, Kizzmekia S. Corbett, Brandon J. DeKosky, Barney S. Graham, John R. Mascola, John Misasi, Tracy Ruckwardt, Nancy J. Sullivan, Lawrence Shapiro, Peter D. Kwong
bioRxiv 2020.06.22.166033; doi: https://doi.org/10.1101/2020.06.22.166033
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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
Tongqing Zhou, I-Ting Teng, Adam S. Olia, Gabriele Cerutti, Jason Gorman, Alexandra Nazzari, Wei Shi, Yaroslav Tsybovsky, Lingshu Wang, Shuishu Wang, Baoshan Zhang, Yi Zhang, Phinikoula S. Katsamba, Yuliya Petrova, Bailey B. Banach, Ahmed S. Fahad, Lihong Liu, Sheila N. Lopez Acevedo, Bharat Madan, Matheus Oliveira de Souza, Xiaoli Pan, Pengfei Wang, Jacy R. Wolfe, Michael Yin, David D. Ho, Emily Phung, Anthony DiPiazza, Lauren Chang, Olubukula Abiona, Kizzmekia S. Corbett, Brandon J. DeKosky, Barney S. Graham, John R. Mascola, John Misasi, Tracy Ruckwardt, Nancy J. Sullivan, Lawrence Shapiro, Peter D. Kwong
bioRxiv 2020.06.22.166033; doi: https://doi.org/10.1101/2020.06.22.166033

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