tetano
Editor, Senior Moderator
Cell Rep
. 2020 Oct 12;108322.
doi: 10.1016/j.celrep.2020.108322. Online ahead of print.
Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes
Tongqing Zhou[SUP] 1 [/SUP], I-Ting Teng[SUP] 1 [/SUP], Adam S Olia[SUP] 1 [/SUP], Gabriele Cerutti[SUP] 2 [/SUP], Jason Gorman[SUP] 1 [/SUP], Alexandra Nazzari[SUP] 1 [/SUP], Wei Shi[SUP] 1 [/SUP], Yaroslav Tsybovsky[SUP] 3 [/SUP], Lingshu Wang[SUP] 1 [/SUP], Shuishu Wang[SUP] 1 [/SUP], Baoshan Zhang[SUP] 1 [/SUP], Yi Zhang[SUP] 1 [/SUP], Phinikoula S Katsamba[SUP] 2 [/SUP], Yuliya Petrova[SUP] 1 [/SUP], Bailey B Banach[SUP] 4 [/SUP], Ahmed S Fahad[SUP] 5 [/SUP], Lihong Liu[SUP] 6 [/SUP], Sheila N Lopez Acevedo[SUP] 5 [/SUP], Bharat Madan[SUP] 5 [/SUP], Matheus Oliveira de Souza[SUP] 5 [/SUP], Xiaoli Pan[SUP] 5 [/SUP], Pengfei Wang[SUP] 6 [/SUP], Jacy R Wolfe[SUP] 5 [/SUP], Michael Yin[SUP] 6 [/SUP], David D Ho[SUP] 6 [/SUP], Emily Phung[SUP] 1 [/SUP], Anthony DiPiazza[SUP] 1 [/SUP], Lauren A Chang[SUP] 1 [/SUP], Olubukola M Abiona[SUP] 1 [/SUP], Kizzmekia S Corbett[SUP] 1 [/SUP], Brandon J DeKosky[SUP] 7 [/SUP], Barney S Graham[SUP] 1 [/SUP], John R Mascola[SUP] 1 [/SUP], John Misasi[SUP] 1 [/SUP], Tracy Ruckwardt[SUP] 1 [/SUP], Nancy J Sullivan[SUP] 1 [/SUP], Lawrence Shapiro[SUP] 8 [/SUP], Peter D Kwong[SUP] 9 [/SUP]
Affiliations
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.
Keywords: COVID-19; HRV3C protease; antibody; biotinylated probe; coronavirus disease 2019; human rhinovirus 3C; single-chain Fc; structure-based design.
. 2020 Oct 12;108322.
doi: 10.1016/j.celrep.2020.108322. Online ahead of print.
Structure-Based Design with Tag-Based Purification and In-Process Biotinylation Enable Streamlined Development of SARS-CoV-2 Spike Molecular Probes
Tongqing Zhou[SUP] 1 [/SUP], I-Ting Teng[SUP] 1 [/SUP], Adam S Olia[SUP] 1 [/SUP], Gabriele Cerutti[SUP] 2 [/SUP], Jason Gorman[SUP] 1 [/SUP], Alexandra Nazzari[SUP] 1 [/SUP], Wei Shi[SUP] 1 [/SUP], Yaroslav Tsybovsky[SUP] 3 [/SUP], Lingshu Wang[SUP] 1 [/SUP], Shuishu Wang[SUP] 1 [/SUP], Baoshan Zhang[SUP] 1 [/SUP], Yi Zhang[SUP] 1 [/SUP], Phinikoula S Katsamba[SUP] 2 [/SUP], Yuliya Petrova[SUP] 1 [/SUP], Bailey B Banach[SUP] 4 [/SUP], Ahmed S Fahad[SUP] 5 [/SUP], Lihong Liu[SUP] 6 [/SUP], Sheila N Lopez Acevedo[SUP] 5 [/SUP], Bharat Madan[SUP] 5 [/SUP], Matheus Oliveira de Souza[SUP] 5 [/SUP], Xiaoli Pan[SUP] 5 [/SUP], Pengfei Wang[SUP] 6 [/SUP], Jacy R Wolfe[SUP] 5 [/SUP], Michael Yin[SUP] 6 [/SUP], David D Ho[SUP] 6 [/SUP], Emily Phung[SUP] 1 [/SUP], Anthony DiPiazza[SUP] 1 [/SUP], Lauren A Chang[SUP] 1 [/SUP], Olubukola M Abiona[SUP] 1 [/SUP], Kizzmekia S Corbett[SUP] 1 [/SUP], Brandon J DeKosky[SUP] 7 [/SUP], Barney S Graham[SUP] 1 [/SUP], John R Mascola[SUP] 1 [/SUP], John Misasi[SUP] 1 [/SUP], Tracy Ruckwardt[SUP] 1 [/SUP], Nancy J Sullivan[SUP] 1 [/SUP], Lawrence Shapiro[SUP] 8 [/SUP], Peter D Kwong[SUP] 9 [/SUP]
Affiliations
- PMID: 33091382
- PMCID: PMC7550166
- DOI: 10.1016/j.celrep.2020.108322
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.
Keywords: COVID-19; HRV3C protease; antibody; biotinylated probe; coronavirus disease 2019; human rhinovirus 3C; single-chain Fc; structure-based design.