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Article: Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody.

Johnson, Nicole V / Wall, Steven C / Kramer, Kevin J / Holt, Clinton M / Periasamy, Sivakumar / Richardson, Simone / Suryadevara, Naveenchandra / Andreano, Emanuele / Paciello, Ida / Pierleoni, Giulio / Piccini, Giulia / Huang, Ying / Ge, Pan / Allen, James D / Uno, Naoko / Shiakolas, Andrea R / Pilewski, Kelsey A / Nargi, Rachel S / Sutton, Rachel E /

Abu-Shmais, Alexandria A / Parks, Robert / Haynes, Barton F / Carnahan, Robert H / Crowe, James E / Montomoli, Emanuele / Rappuoli, Rino / Bukreyev, Alexander / Ross, Ted M / Sautto, Giuseppe A / McLellan, Jason S / Georgiev, Ivelin S

bioRxiv : the preprint server for biology

2024

Abstract: Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic ... ...

Abstract	Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.
Language	English
Publishing date	2024-01-16
Publishing country	United States
Document type	Preprint
DOI	10.1101/2024.01.15.575741
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Article ; Online: Defects in Efflux (

Shen, Xiaoyu / Johnson, Nicole V / Kreamer, Naomi N K / Barnes, S Whitney / Walker, John R / Woods, Angela L / Six, David A / Dean, C R

Antimicrobial agents and chemotherapy

2019 Volume 63, Issue 7

Abstract: Antibiotic hypersensitive bacterial mutants (e.g., ...

Abstract	Antibiotic hypersensitive bacterial mutants (e.g.,
MeSH term(s)	Anti-Bacterial Agents/pharmacology ; Bacterial Outer Membrane Proteins/genetics ; Bacterial Proteins/genetics ; Biological Transport/genetics ; Cell Membrane Permeability/genetics ; Lipopolysaccharides/metabolism ; Membrane Transport Proteins/genetics ; Microbial Sensitivity Tests/methods ; Mutation/genetics ; Pseudomonas aeruginosa/drug effects ; Pseudomonas aeruginosa/genetics ; beta-Lactamases/genetics ; beta-Lactams/pharmacology
Chemical Substances	Anti-Bacterial Agents ; Bacterial Outer Membrane Proteins ; Bacterial Proteins ; Lipopolysaccharides ; Membrane Transport Proteins ; beta-Lactams ; AmpC beta-lactamases (EC 3.5.2.6) ; beta-Lactamases (EC 3.5.2.6)
Language	English
Publishing date	2019-06-24
Publishing country	United States
Document type	Journal Article
ZDB-ID	217602-6
ISSN	1098-6596 ; 0066-4804
ISSN (online)	1098-6596
ISSN	0066-4804
DOI	10.1128/AAC.00784-19
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Article ; Online: Epigenetic cell fate in Candida albicans is controlled by transcription factor condensates acting at super-enhancer-like elements.

Frazer, Corey / Staples, Mae I / Kim, Yoori / Hirakawa, Matthew / Dowell, Maureen A / Johnson, Nicole V / Hernday, Aaron D / Ryan, Veronica H / Fawzi, Nicolas L / Finkelstein, Ilya J / Bennett, Richard J

Nature microbiology

2020 Volume 5, Issue 11, Page(s) 1374–1389

Abstract: Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching ... ...

Abstract	Cell identity in eukaryotes is controlled by transcriptional regulatory networks that define cell-type-specific gene expression. In the opportunistic fungal pathogen Candida albicans, transcriptional regulatory networks regulate epigenetic switching between two alternative cell states, 'white' and 'opaque', that exhibit distinct host interactions. In the present study, we reveal that the transcription factors (TFs) regulating cell identity contain prion-like domains (PrLDs) that enable liquid-liquid demixing and the formation of phase-separated condensates. Multiple white-opaque TFs can co-assemble into complex condensates as observed on single DNA molecules. Moreover, heterotypic interactions between PrLDs support the assembly of multifactorial condensates at a synthetic locus within live eukaryotic cells. Mutation of the Wor1 TF revealed that substitution of acidic residues in the PrLD blocked its ability to phase separate and co-recruit other TFs in live cells, as well as its function in C. albicans cell fate determination. Together, these studies reveal that PrLDs support the assembly of TF complexes that control fungal cell identity and highlight parallels with the 'super-enhancers' that regulate mammalian cell fate.
MeSH term(s)	Candida albicans/cytology ; Candida albicans/genetics ; Cell Line, Tumor ; DNA, Fungal/genetics ; DNA, Fungal/metabolism ; Enhancer Elements, Genetic ; Epigenesis, Genetic ; Fungal Proteins/chemistry ; Fungal Proteins/genetics ; Fungal Proteins/metabolism ; Gene Expression Regulation, Fungal ; Gene Regulatory Networks ; Humans ; Mutation ; Phenotype ; Prions/chemistry ; Protein Aggregates ; Protein Domains ; Transcription Factors/chemistry ; Transcription Factors/genetics ; Transcription Factors/metabolism
Chemical Substances	DNA, Fungal ; Fungal Proteins ; Prions ; Protein Aggregates ; Transcription Factors
Language	English
Publishing date	2020-07-27
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	2058-5276
ISSN (online)	2058-5276
DOI	10.1038/s41564-020-0760-7
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Article ; Online: Massively parallel kinetic profiling of natural and engineered CRISPR nucleases.

Jones, Stephen K / Hawkins, John A / Johnson, Nicole V / Jung, Cheulhee / Hu, Kuang / Rybarski, James R / Chen, Janice S / Doudna, Jennifer A / Press, William H / Finkelstein, Ilya J

Nature biotechnology

2020 Volume 39, Issue 1, Page(s) 84–93

Abstract: Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq- ...

Abstract	Engineered SpCas9s and AsCas12a cleave fewer off-target genomic sites than wild-type (wt) Cas9. However, understanding their fidelity, mechanisms and cleavage outcomes requires systematic profiling across mispaired target DNAs. Here we describe NucleaSeq-nuclease digestion and deep sequencing-a massively parallel platform that measures the cleavage kinetics and time-resolved cleavage products for over 10,000 targets containing mismatches, insertions and deletions relative to the guide RNA. Combining cleavage rates and binding specificities on the same target libraries, we benchmarked five SpCas9 variants and AsCas12a. A biophysical model built from these data sets revealed mechanistic insights into off-target cleavage. Engineered Cas9s, especially Cas9-HF1, dramatically increased cleavage specificity but not binding specificity compared to wtCas9. Surprisingly, AsCas12a cleavage specificity differed little from that of wtCas9. Initial DNA cleavage sites and end trimming varied by nuclease, guide RNA and the positions of mispaired nucleotides. More broadly, NucleaSeq enables rapid, quantitative and systematic comparisons of specificity and cleavage outcomes across engineered and natural nucleases.
MeSH term(s)	Bacterial Proteins/chemistry ; Bacterial Proteins/genetics ; Bacterial Proteins/metabolism ; CRISPR-Associated Protein 9/chemistry ; CRISPR-Associated Protein 9/genetics ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/chemistry ; CRISPR-Associated Proteins/genetics ; CRISPR-Associated Proteins/metabolism ; CRISPR-Cas Systems ; Endodeoxyribonucleases/chemistry ; Endodeoxyribonucleases/genetics ; Endodeoxyribonucleases/metabolism ; Gene Editing ; High-Throughput Nucleotide Sequencing/methods ; Kinetics ; Protein Binding/genetics ; Protein Engineering ; RNA, Guide, CRISPR-Cas Systems/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Substrate Specificity/genetics
Chemical Substances	Bacterial Proteins ; CRISPR-Associated Proteins ; RNA, Guide, CRISPR-Cas Systems ; CRISPR-Associated Protein 9 (EC 3.1.-) ; Cas12a protein (EC 3.1.-) ; Endodeoxyribonucleases (EC 3.1.-)
Language	English
Publishing date	2020-09-07
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-020-0646-5
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Article ; Online: SARS-CoV-2 escape from a highly neutralizing COVID-19 convalescent plasma.

Andreano, Emanuele / Piccini, Giulia / Licastro, Danilo / Casalino, Lorenzo / Johnson, Nicole V / Paciello, Ida / Dal Monego, Simeone / Pantano, Elisa / Manganaro, Noemi / Manenti, Alessandro / Manna, Rachele / Casa, Elisa / Hyseni, Inesa / Benincasa, Linda / Montomoli, Emanuele / Amaro, Rommie E / McLellan, Jason S / Rappuoli, Rino

Proceedings of the National Academy of Sciences of the United States of America

2021 Volume 118, Issue 36

Abstract: To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully ... ...

Abstract	To investigate the evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the immune population, we coincupi bated the authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for seven passages, but, after 45 d, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed, at day 80, by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom, South Africa, Brazil, and Japan of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed.
MeSH term(s)	Amino Acid Substitution ; Angiotensin-Converting Enzyme 2/chemistry ; Angiotensin-Converting Enzyme 2/genetics ; Angiotensin-Converting Enzyme 2/immunology ; Animals ; Antibodies, Neutralizing/chemistry ; Antibodies, Neutralizing/genetics ; Antibodies, Neutralizing/immunology ; Antibodies, Neutralizing/pharmacology ; Antibodies, Viral/chemistry ; Antibodies, Viral/genetics ; Antibodies, Viral/immunology ; Antibodies, Viral/pharmacology ; Binding Sites ; COVID-19/genetics ; COVID-19/immunology ; COVID-19/virology ; Chlorocebus aethiops ; Convalescence ; Gene Expression ; Humans ; Immune Evasion ; Immune Sera/chemistry ; Models, Molecular ; Mutation ; Neutralization Tests ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; SARS-CoV-2/drug effects ; SARS-CoV-2/genetics ; SARS-CoV-2/immunology ; SARS-CoV-2/pathogenicity ; Spike Glycoprotein, Coronavirus/chemistry ; Spike Glycoprotein, Coronavirus/genetics ; Spike Glycoprotein, Coronavirus/immunology ; Vero Cells
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Immune Sera ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2 ; ACE2 protein, human (EC 3.4.17.23) ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2021-08-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2103154118
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Article ; Online: Efficient discovery of SARS-CoV-2-neutralizing antibodies via B cell receptor sequencing and ligand blocking.

Shiakolas, Andrea R / Kramer, Kevin J / Johnson, Nicole V / Wall, Steven C / Suryadevara, Naveenchandra / Wrapp, Daniel / Periasamy, Sivakumar / Pilewski, Kelsey A / Raju, Nagarajan / Nargi, Rachel / Sutton, Rachel E / Walker, Lauren M / Setliff, Ian / Crowe, James E / Bukreyev, Alexander / Carnahan, Robert H / McLellan, Jason S / Georgiev, Ivelin S

Nature biotechnology

2022 Volume 40, Issue 8, Page(s) 1270–1275

Abstract: Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often ... ...

Abstract	Although several monoclonal antibodies (mAbs) targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been approved for coronavirus disease 2019 (COVID-19) therapy, development was generally inefficient, with lead generation often requiring the production and testing of numerous antibody candidates. Here, we report that the integration of target-ligand blocking with a previously described B cell receptor-sequencing approach (linking B cell receptor to antigen specificity through sequencing (LIBRA-seq)) enables the rapid and efficient identification of multiple neutralizing mAbs that prevent the binding of SARS-CoV-2 spike (S) protein to angiotensin-converting enzyme 2 (ACE2). The combination of target-ligand blocking and high-throughput antibody sequencing promises to increase the throughput of programs aimed at discovering new neutralizing antibodies.
MeSH term(s)	Antibodies, Neutralizing/genetics ; Antibodies, Neutralizing/therapeutic use ; Antibodies, Viral/genetics ; Antibodies, Viral/therapeutic use ; COVID-19 ; Humans ; Ligands ; Peptidyl-Dipeptidase A ; Receptors, Antigen, B-Cell/genetics ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus
Chemical Substances	Antibodies, Neutralizing ; Antibodies, Viral ; Ligands ; Receptors, Antigen, B-Cell ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2 ; Peptidyl-Dipeptidase A (EC 3.4.15.1)
Language	English
Publishing date	2022-03-03
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	1311932-1
ISSN	1546-1696 ; 1087-0156
ISSN (online)	1546-1696
ISSN	1087-0156
DOI	10.1038/s41587-022-01232-2
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Article ; Online: Discovery and Characterization of a Pan-betacoronavirus S2-binding antibody

Johnson, Nicole V. / Wall, Steven C. / Kramer, Kevin J. / Holt, Clinton M. / Periasamy, Sivakumar / Richardson, Simone / Suryadevara, Naveenchandra / Andreano, Emanuele / Paciello, Ida / Pierleoni, Giulio / Piccini, Giulia / Huang, Ying / Ge, Pan / Allen, James D. / Uno, Naoko / Shiakolas, Andrea R. / Pilewski, Kelsey A. / Nargi, Rachel S. / Sutton, Rachel E. /

Abu-Shmais, Alexandria A. / Parks, Robert / Haynes, Barton F. / Carnahan, Robert H. / Crowe, James E. / Montomoli, Emanuele / Rappuoli, Rino / Bukreyev, Alexander / Ross, Ted M. / Sautto, Giuseppe A. / McLellan, Jason S. / Georgiev, Ivelin S.

bioRxiv

Abstract	Three coronaviruses have spilled over from animal reservoirs into the human population and caused deadly epidemics or pandemics. The continued emergence of coronaviruses highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using LIBRA-seq, we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these antibodies, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryo-EM structure of 54043-5 bound to the pre-fusion S2 subunit of the SARS-CoV-2 spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses, including ADCC and ADCP. In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.
Keywords	covid19
Language	English
Publishing date	2024-01-16
Publisher	Cold Spring Harbor Laboratory
Document type	Article ; Online
DOI	10.1101/2024.01.15.575741
Database	COVID19

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Article: SARS-CoV-2 escape

bioRxiv : the preprint server for biology

2020

Abstract: To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the ... ...

Abstract	To investigate the evolution of SARS-CoV-2 in the immune population, we co-incubated authentic virus with a highly neutralizing plasma from a COVID-19 convalescent patient. The plasma fully neutralized the virus for 7 passages, but after 45 days, the deletion of F140 in the spike N-terminal domain (NTD) N3 loop led to partial breakthrough. At day 73, an E484K substitution in the receptor-binding domain (RBD) occurred, followed at day 80 by an insertion in the NTD N5 loop containing a new glycan sequon, which generated a variant completely resistant to plasma neutralization. Computational modeling predicts that the deletion and insertion in loops N3 and N5 prevent binding of neutralizing antibodies. The recent emergence in the United Kingdom and South Africa of natural variants with similar changes suggests that SARS-CoV-2 has the potential to escape an effective immune response and that vaccines and antibodies able to control emerging variants should be developed. One sentence summary: Three mutations allowed SARS-CoV-2 to evade the polyclonal antibody response of a highly neutralizing COVID-19 convalescent plasma.
Language	English
Publishing date	2020-12-28
Publishing country	United States
Document type	Preprint
DOI	10.1101/2020.12.28.424451
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Article ; Online: Potent neutralization of SARS-CoV-2 variants of concern by an antibody with an uncommon genetic signature and structural mode of spike recognition.

Kramer, Kevin J / Johnson, Nicole V / Shiakolas, Andrea R / Suryadevara, Naveenchandra / Periasamy, Sivakumar / Raju, Nagarajan / Williams, Jazmean K / Wrapp, Daniel / Zost, Seth J / Walker, Lauren M / Wall, Steven C / Holt, Clinton M / Hsieh, Ching-Lin / Sutton, Rachel E / Paulo, Ariana / Nargi, Rachel S / Davidson, Edgar / Doranz, Benjamin J / Crowe, James E /

Bukreyev, Alexander / Carnahan, Robert H / McLellan, Jason S / Georgiev, Ivelin S

Cell reports

2021 Volume 37, Issue 1, Page(s) 109784

Abstract: The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease ( ... ...

Abstract	The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages that are more transmissible and resistant to currently approved antibody therapies poses a considerable challenge to the clinical treatment of coronavirus disease (COVID-19). Therefore, the need for ongoing discovery efforts to identify broadly reactive monoclonal antibodies to SARS-CoV-2 is of utmost importance. Here, we report a panel of SARS-CoV-2 antibodies isolated using the linking B cell receptor to antigen specificity through sequencing (LIBRA-seq) technology from an individual who recovered from COVID-19. Of these antibodies, 54042-4 shows potent neutralization against authentic SARS-CoV-2 viruses, including variants of concern (VOCs). A cryoelectron microscopy (cryo-EM) structure of 54042-4 in complex with the SARS-CoV-2 spike reveals an epitope composed of residues that are highly conserved in currently circulating SARS-CoV-2 lineages. Further, 54042-4 possesses uncommon genetic and structural characteristics that distinguish it from other potently neutralizing SARS-CoV-2 antibodies. Together, these findings provide motivation for the development of 54042-4 as a lead candidate to counteract current and future SARS-CoV-2 VOCs.
MeSH term(s)	Angiotensin-Converting Enzyme 2/chemistry ; Angiotensin-Converting Enzyme 2/immunology ; Animals ; Antibodies, Monoclonal/immunology ; Antibodies, Neutralizing/immunology ; Antibodies, Viral/immunology ; Antibody Formation ; COVID-19/genetics ; COVID-19/immunology ; COVID-19/virology ; Cell Line ; Chlorocebus aethiops ; Cryoelectron Microscopy ; Epitope Mapping/methods ; Epitopes/chemistry ; Epitopes/immunology ; High-Throughput Screening Assays/methods ; Humans ; Male ; Middle Aged ; Protein Binding ; Protein Interaction Domains and Motifs ; Receptors, Antigen, B-Cell/chemistry ; Receptors, Antigen, B-Cell/immunology ; SARS-CoV-2/chemistry ; SARS-CoV-2/genetics ; SARS-CoV-2/immunology ; Spike Glycoprotein, Coronavirus/chemistry ; Spike Glycoprotein, Coronavirus/immunology ; Vero Cells
Chemical Substances	Antibodies, Monoclonal ; Antibodies, Neutralizing ; Antibodies, Viral ; Epitopes ; Receptors, Antigen, B-Cell ; Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2 ; ACE2 protein, human (EC 3.4.17.23) ; Angiotensin-Converting Enzyme 2 (EC 3.4.17.23)
Language	English
Publishing date	2021-09-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2649101-1
ISSN	2211-1247 ; 2211-1247
ISSN (online)	2211-1247
ISSN	2211-1247
DOI	10.1016/j.celrep.2021.109784
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Article ; Online: Expression and characterization of SARS-CoV-2 spike proteins.

Nature protocols

2021 Volume 16, Issue 11, Page(s) 5339–5356

Abstract: The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it ... ...

Abstract	The severe acute respiratory syndrome coronavirus 2 spike protein is a critical component of coronavirus disease 2019 vaccines and diagnostics and is also a therapeutic target. However, the spike protein is difficult to produce recombinantly because it is a large trimeric class I fusion membrane protein that is metastable and heavily glycosylated. We recently developed a prefusion-stabilized spike variant, termed HexaPro for six stabilizing proline substitutions, that can be expressed with a yield of >30 mg/L in ExpiCHO cells. This protocol describes an optimized workflow for expressing and biophysically characterizing rationally engineered spike proteins in Freestyle 293 and ExpiCHO cell lines. Although we focus on HexaPro, this protocol has been used to purify over a hundred different spike variants in our laboratories. We also provide guidance on expression quality control, long-term storage, and uses in enzyme-linked immunosorbent assays. The entire protocol, from transfection to biophysical characterization, can be completed in 7 d by researchers with basic tissue cell culture and protein purification expertise.
MeSH term(s)	Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Gene Expression Regulation, Viral/physiology ; HEK293 Cells ; Humans ; Models, Molecular ; Protein Conformation ; SARS-CoV-2 ; Spike Glycoprotein, Coronavirus/genetics ; Spike Glycoprotein, Coronavirus/metabolism
Chemical Substances	Spike Glycoprotein, Coronavirus ; spike protein, SARS-CoV-2
Language	English
Publishing date	2021-10-05
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2244966-8
ISSN	1750-2799 ; 1754-2189
ISSN (online)	1750-2799
ISSN	1754-2189
DOI	10.1038/s41596-021-00623-0
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