Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface
The interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus–cell interface making it the main target of neutralizing antibodies. Here, we...
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| Eará dahkkit: | , , , , , , , |
| Materiálatiipa: | article |
| Giella: | eaŋgalasgiella |
| Almmustuhtton: |
2023
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| Fáttát: | |
| Liŋkkat: | https://hdl.handle.net/20.500.12381/3854 |
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| _version_ | 1868890177577943040 |
|---|---|
| author | Carrión, Federico |
| author2 | Rammauro, Florencia Olivero, Natalia Fló, Martín Portela, María Magdalena Lima, Analía Durán, Rosario Pristch, Otto Bianchi, Sergio |
| author2_role | author author author author author author author author |
| author_browse | Bianchi, Sergio Carrión, Federico Durán, Rosario Fló, Martín Lima, Analía Olivero, Natalia Portela, María Magdalena Pristch, Otto Rammauro, Florencia |
| author_facet | Carrión, Federico Rammauro, Florencia Olivero, Natalia Fló, Martín Portela, María Magdalena Lima, Analía Durán, Rosario Pristch, Otto Bianchi, Sergio |
| author_role | author |
| collection | IPMON en REDI |
| dc.creator.none.fl_str_mv | Carrión, Federico Rammauro, Florencia Olivero, Natalia Fló, Martín Portela, María Magdalena Lima, Analía Durán, Rosario Pristch, Otto Bianchi, Sergio |
| dc.date.none.fl_str_mv | 2023-07-03 2025-02-07T13:54:55Z 2025-02-07T13:54:55Z |
| dc.identifier.none.fl_str_mv | https://hdl.handle.net/20.500.12381/3854 FMV_3_2022_1_172395 10.1002/pro.4721 |
| dc.language.none.fl_str_mv | eng |
| dc.publisher.none.fl_str_mv | Wiley |
| dc.rights.none.fl_str_mv | Acceso abierto info:eu-repo/semantics/openAccess Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional. (CC BY-NC-ND) |
| dc.source.none.fl_str_mv | Protein Science reponame:IPMON en REDI instname:Institut Pasteur de Montevideo instacron:Institut Pasteur de Montevideo |
| dc.subject.none.fl_str_mv | ACE2, binding, Drosophila, microheterogeneity, neutralization, RBD, S2 cells, SARS-CoV-2, virus–cell interface Ciencias Naturales y Exactas Ciencias Biológicas Virología |
| dc.title.none.fl_str_mv | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| dc.type.none.fl_str_mv | Artículo info:eu-repo/semantics/article Publicado info:eu-repo/semantics/publishedVersion |
| description | The interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus–cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost-effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep-tag with yields >40 mg L−1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep-tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep-Tactin-HRP as a proof-of-concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in-house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | anni_bc39e2f6d7fd362287a2c9040963e09e |
| identifier_str_mv | FMV_3_2022_1_172395 10.1002/pro.4721 |
| instacron_str | Institut Pasteur de Montevideo |
| institution | Institut Pasteur de Montevideo |
| instname_str | Institut Pasteur de Montevideo |
| language | eng |
| network_acronym_str | anni |
| network_name_str | oai-lr-anni |
| oai_identifier_str | oai:redi.anii.org.uy:20.500.12381/3854 |
| publishDate | 2023 |
| publishDateSort | 2023 |
| publisher.none.fl_str_mv | Wiley |
| reponame_str | IPMON en REDI |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | Acceso abierto Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional. (CC BY-NC-ND) |
| spelling | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interfaceCarrión, FedericoRammauro, FlorenciaOlivero, NataliaFló, MartínPortela, María MagdalenaLima, AnalíaDurán, RosarioPristch, OttoBianchi, SergioACE2, binding, Drosophila, microheterogeneity, neutralization, RBD, S2 cells, SARS-CoV-2, virus–cell interfaceCiencias Naturales y ExactasCiencias BiológicasVirologíaThe interaction between the receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 and the peptidase domain of the human angiotensin-converting enzyme 2 (ACE2) allows the first specific contact at the virus–cell interface making it the main target of neutralizing antibodies. Here, we show a unique and cost-effective protocol using Drosophila S2 cells to produce both RBD and soluble human ACE2 peptidase domain (shACE2) as thermostable proteins, purified via Strep-tag with yields >40 mg L−1 in a laboratory scale. Furthermore, we demonstrate its binding with KD values in the lower nanomolar range (independently of Strep-tag removal) and its capability to be blocked by serum antibodies in a competition ELISA with Strep-Tactin-HRP as a proof-of-concept. In addition, we assess the capacity of RBD to bind native dimeric ACE2 overexpressed in human cells and its antigen properties with specific serum antibodies. Finally, for completeness, we analyzed RBD microheterogeneity associated with glycosylation and negative charges, with negligible effect on binding either with antibodies or shACE2. Our system represents an accessible and reliable tool for designing in-house surrogate virus neutralization tests (sVNTs), enabling the rapid characterization of neutralizing humoral responses elicited against vaccines or infection, especially in the absence of facilities to conduct virus neutralization tests. Moreover, our biophysical and biochemical characterization of RBD and shACE2 produced in S2 cells lays the groundwork for adapting to different variants of concern (VOCs) to study humoral responses elicited against different VOCs and vaccine formulations.Wiley2025-02-07T13:54:55Z2025-02-07T13:54:55Z2023-07-03Artículoinfo:eu-repo/semantics/articlePublicadoinfo:eu-repo/semantics/publishedVersionhttps://hdl.handle.net/20.500.12381/3854FMV_3_2022_1_17239510.1002/pro.4721Protein Sciencereponame:IPMON en REDIinstname:Institut Pasteur de Montevideoinstacron:Institut Pasteur de MontevideoengAcceso abiertoinfo:eu-repo/semantics/openAccessReconocimiento-NoComercial-SinObraDerivada 4.0 Internacional. (CC BY-NC-ND)oai:redi.anii.org.uy:20.500.12381/38542026-06-16T05:20:19Z |
| spellingShingle | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface Carrión, Federico ACE2, binding, Drosophila, microheterogeneity, neutralization, RBD, S2 cells, SARS-CoV-2, virus–cell interface Ciencias Naturales y Exactas Ciencias Biológicas Virología |
| status_str | publishedVersion |
| title | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| title_full | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| title_fullStr | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| title_full_unstemmed | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| title_short | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| title_sort | Soluble SARS-CoV-2 RBD and human ACE2 peptidase domain produced in Drosophila S2 cells show functions evoking virus–cell interface |
| topic | ACE2, binding, Drosophila, microheterogeneity, neutralization, RBD, S2 cells, SARS-CoV-2, virus–cell interface Ciencias Naturales y Exactas Ciencias Biológicas Virología |
| url | https://hdl.handle.net/20.500.12381/3854 |