A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System
Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-03-01
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| Series: | Bioengineering |
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| Online Access: | https://www.mdpi.com/2306-5354/10/3/389 |
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| author | Dóra Nagy-Fazekas Pál Stráner Péter Ecsédi Nóra Taricska Adina Borbély László Nyitray András Perczel |
| author_facet | Dóra Nagy-Fazekas Pál Stráner Péter Ecsédi Nóra Taricska Adina Borbély László Nyitray András Perczel |
| author_sort | Dóra Nagy-Fazekas |
| collection | DOAJ |
| description | Antibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an <i>Escherichia coli</i> protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a K<sub>d</sub> of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants. |
| first_indexed | 2024-03-11T06:55:24Z |
| format | Article |
| id | doaj.art-532fcbcce04c4c5eaab7d71a9c0ff521 |
| institution | Directory Open Access Journal |
| issn | 2306-5354 |
| language | English |
| last_indexed | 2024-03-11T06:55:24Z |
| publishDate | 2023-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Bioengineering |
| spelling | doaj.art-532fcbcce04c4c5eaab7d71a9c0ff5212023-11-17T09:40:37ZengMDPI AGBioengineering2306-53542023-03-0110338910.3390/bioengineering10030389A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial SystemDóra Nagy-Fazekas0Pál Stráner1Péter Ecsédi2Nóra Taricska3Adina Borbély4László Nyitray5András Perczel6Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, HungaryDepartment of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, HungaryMTA-ELTE Lendület Ion Mobility Mass Spectrometry Research Group, Department of Analytical Chemistry, ELTE Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, HungaryDepartment of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/C, H-1117 Budapest, HungaryLaboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány Péter sétány 1/A, H-1117 Budapest, HungaryAntibodies are key proteins of the immune system, and they are widely used for both research and theragnostic applications. Among them, camelid immunoglobulins (IgG) differ from the canonical human IgG molecules, as their light chains are completely missing; thus, they have only variable domains on their heavy chains (VHHs). A single VHH domain, often called a nanobody, has favorable structural, biophysical, and functional features compared to canonical antibodies. Therefore, robust and efficient production protocols relying on recombinant technologies are in high demand. Here, by utilizing ecotin, an <i>Escherichia coli</i> protein, as a fusion partner, we present a bacterial expression system that allows an easy, fast, and cost-effective way to prepare nanobodies. Ecotin was used here as a periplasmic translocator and a passive refolding chaperone, which allowed us to reach high-yield production of nanobodies. We also present a new, easily applicable prokaryotic expression and purification method of the receptor-binding domain (RBD) of the SARS-CoV-2 S protein for interaction assays. We demonstrate using ECD spectroscopy that the bacterially produced RBD is well-folded. The bacterially produced nanobody was shown to bind strongly to the recombinant RBD, with a K<sub>d</sub> of 10 nM. The simple methods presented here could facilitate rapid interaction measurements in the event of the appearance of additional SARS-CoV-2 variants.https://www.mdpi.com/2306-5354/10/3/389camelid immunoglobulinsnanobodybacterial expressionwell-folded SARS-CoV-2 S protein RBD variants |
| spellingShingle | Dóra Nagy-Fazekas Pál Stráner Péter Ecsédi Nóra Taricska Adina Borbély László Nyitray András Perczel A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System Bioengineering camelid immunoglobulins nanobody bacterial expression well-folded SARS-CoV-2 S protein RBD variants |
| title | A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System |
| title_full | A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System |
| title_fullStr | A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System |
| title_full_unstemmed | A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System |
| title_short | A Novel Fusion Protein System for the Production of Nanobodies and the SARS-CoV-2 Spike RBD in a Bacterial System |
| title_sort | novel fusion protein system for the production of nanobodies and the sars cov 2 spike rbd in a bacterial system |
| topic | camelid immunoglobulins nanobody bacterial expression well-folded SARS-CoV-2 S protein RBD variants |
| url | https://www.mdpi.com/2306-5354/10/3/389 |
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