Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i>
Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial chall...
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MDPI AG
2025-01-01
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| Online Access: | https://www.mdpi.com/2218-273X/15/1/111 |
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| author | Shuai Zhao Wanting Zeng Fang Yu Pingping Xu Chin-Yu Chen Wanping Chen Yanming Dong Fei Wang Lixin Ma |
| author_facet | Shuai Zhao Wanting Zeng Fang Yu Pingping Xu Chin-Yu Chen Wanping Chen Yanming Dong Fei Wang Lixin Ma |
| author_sort | Shuai Zhao |
| collection | DOAJ |
| description | Nanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with <i>Escherichia coli</i> by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of <i>E. coli</i> cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by <i>E. coli</i> retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, <i>E. coli</i> cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones. |
| format | Article |
| id | doaj-art-0b4cf52164bf48a0a9a2db918faf12d4 |
| institution | Kabale University |
| issn | 2218-273X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj-art-0b4cf52164bf48a0a9a2db918faf12d42025-01-24T13:25:13ZengMDPI AGBiomolecules2218-273X2025-01-0115111110.3390/biom15010111Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i>Shuai Zhao0Wanting Zeng1Fang Yu2Pingping Xu3Chin-Yu Chen4Wanping Chen5Yanming Dong6Fei Wang7Lixin Ma8State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaState Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, ChinaNanobodies have gained attention as potential therapeutic and diagnostic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due to their ability to bind and neutralize the virus. However, rapid, scalable, and robust production of nanobodies for SARS-CoV-2 remains a crucial challenge. In this study, we developed a visual and high-efficiency biomanufacturing method for nanobodies with <i>Escherichia coli</i> by fusing the super-folder green fluorescent protein (sfGFP) to the N-terminus or C-terminus of the nanobody. Several receptor-binding domain (RBD)-specific nanobodies of the SARS-CoV-2 spike protein (S) were secreted onto the surface of <i>E. coli</i> cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by <i>E. coli</i> retained equal activity as prior research, regardless of whether sfGFP was removed. Since some of the nanobodies bound to different regions of the RBD, we combined two nanobodies to improve the affinity. Fu2-sfGFP-ANTE was constructed to be bispecific for the RBD, and the bispecific nanobody exhibited significantly higher affinity than Fu2 (35.0-fold), ANTE (7.3-fold), and the combination of the two nanobodies (3.3-fold). Notably, Fu2-sfGFP-ANTE can be normally secreted into the culture medium and outer membrane. The novel nanobody production system enhances the efficiency of nanobody expression and streamlines the downstream purification process, enabling large-scale, cost-effective nanobody production. In addition, <i>E. coli</i> cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones.https://www.mdpi.com/2218-273X/15/1/111nanobodySARS-CoV-2sfGFPsecretory expressionbispecific nanobody |
| spellingShingle | Shuai Zhao Wanting Zeng Fang Yu Pingping Xu Chin-Yu Chen Wanping Chen Yanming Dong Fei Wang Lixin Ma Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> Biomolecules nanobody SARS-CoV-2 sfGFP secretory expression bispecific nanobody |
| title | Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> |
| title_full | Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> |
| title_fullStr | Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> |
| title_full_unstemmed | Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> |
| title_short | Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with <i>Escherichia coli</i> |
| title_sort | visual and high efficiency secretion of sars cov 2 nanobodies with i escherichia coli i |
| topic | nanobody SARS-CoV-2 sfGFP secretory expression bispecific nanobody |
| url | https://www.mdpi.com/2218-273X/15/1/111 |
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