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Science Foundation of Wuhan City","award":["2024BCA001"],"award-info":[{"award-number":["2024BCA001"]}]},{"name":"Project of Technological Innovation Plan in Hubei Province","award":["32070171"],"award-info":[{"award-number":["32070171"]}]},{"name":"Project of Technological Innovation Plan in Hubei Province","award":["2023AFB243"],"award-info":[{"award-number":["2023AFB243"]}]},{"name":"Project of Technological Innovation Plan in Hubei Province","award":["2023020302020708"],"award-info":[{"award-number":["2023020302020708"]}]},{"name":"Project of Technological Innovation Plan in Hubei Province","award":["2024040701010046"],"award-info":[{"award-number":["2024040701010046"]}]},{"name":"Project of Technological Innovation Plan in Hubei Province","award":["2024BCA001"],"award-info":[{"award-number":["2024BCA001"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>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 Escherichia coli 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 E. coli cells and even into the culture medium, including Fu2, ANTE, mNb6, MR3-MR3, and n3113.1. The nanobodies secreted by E. coli 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, E. coli cells secreting the nanobodies on their surface facilitates screening and characterization of antigen-binding clones.<\/jats:p>","DOI":"10.3390\/biom15010111","type":"journal-article","created":{"date-parts":[[2025,1,13]],"date-time":"2025-01-13T04:01:52Z","timestamp":1736740912000},"page":"111","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Visual and High-Efficiency Secretion of SARS-CoV-2 Nanobodies with Escherichia coli"],"prefix":"10.3390","volume":"15","author":[{"given":"Shuai","family":"Zhao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanting","family":"Zeng","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Yu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pingping","family":"Xu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chin-Yu","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanping","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5756-5625","authenticated-orcid":false,"given":"Yanming","family":"Dong","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fei","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4938-4741","authenticated-orcid":false,"given":"Lixin","family":"Ma","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"860","DOI":"10.1126\/science.abb5793","article-title":"Projecting the transmission dynamics of SARS-CoV-2 through the postpandemic period","volume":"368","author":"Kissler","year":"2020","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"265","DOI":"10.1038\/s41586-020-2008-3","article-title":"A new coronavirus associated with human respiratory disease in China","volume":"579","author":"Wu","year":"2020","journal-title":"Nature"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"758","DOI":"10.1038\/s41586-022-05542-y","article-title":"SARS-CoV-2 infection and persistence in the human body and brain at autopsy","volume":"612","author":"Stein","year":"2022","journal-title":"Nature"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"685","DOI":"10.1038\/s41579-021-00630-8","article-title":"Structural biology of SARS-CoV-2 and implications for therapeutic development","volume":"19","author":"Yang","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"2097","DOI":"10.1056\/NEJMoa2202261","article-title":"Efficacy and Safety of the RBD-Dimer\u2013Based Covid-19 Vaccine ZF2001 in Adults","volume":"386","author":"Dai","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2265","DOI":"10.1016\/j.cell.2022.04.029","article-title":"Protective prototype-Beta and Delta-Omicron chimeric RBD-dimer vaccines against SARS-CoV-2","volume":"185","author":"Xu","year":"2022","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"311","DOI":"10.1038\/s41467-023-35949-8","article-title":"Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2","volume":"14","author":"Huang","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1038\/s41586-020-2381-y","article-title":"A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2","volume":"584","author":"Shi","year":"2020","journal-title":"Nature"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"112","DOI":"10.1038\/s41579-022-00809-7","article-title":"SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies","volume":"21","author":"Cox","year":"2022","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1038\/s41586-021-03676-z","article-title":"Nanobodies from camelid mice and llamas neutralize SARS-CoV-2 variants","volume":"595","author":"Xu","year":"2021","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"466","DOI":"10.1111\/imr.13385","article-title":"Nanobody-based heavy chain antibodies and chimeric antibodies","volume":"328","year":"2024","journal-title":"Immunol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"5920","DOI":"10.1038\/s41467-023-41631-w","article-title":"Camel nanobody-based B7-H3 CAR-T cells show high efficacy against large solid tumours","volume":"14","author":"Li","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"105645","DOI":"10.1016\/j.pep.2020.105645","article-title":"Recombinant expression of nanobodies and nanobody-derived immunoreagents","volume":"172","year":"2020","journal-title":"Protein Expr. Purif."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1038\/s41594-018-0028-6","article-title":"Yeast surface display platform for rapid discovery of conformationally selective nanobodies","volume":"25","author":"McMahon","year":"2018","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5588","DOI":"10.1038\/s41467-020-19204-y","article-title":"Selection, biophysical and structural analysis of synthetic nanobodies that effectively neutralize SARS-CoV-2","volume":"11","author":"Custodio","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"4635","DOI":"10.1038\/s41467-021-24905-z","article-title":"A synthetic nanobody targeting RBD protects hamsters from SARS-CoV-2 infection","volume":"12","author":"Li","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"155","DOI":"10.1038\/s41467-021-27610-z","article-title":"A bispecific monomeric nanobody induces spike trimer dimers and neutralizes SARS-CoV-2 in vivo","volume":"13","author":"Hanke","year":"2022","journal-title":"Nat. Commun."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1473","DOI":"10.1126\/science.abe3255","article-title":"An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike","volume":"370","author":"Schoof","year":"2020","journal-title":"Science"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1479","DOI":"10.1126\/science.abe4747","article-title":"Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2","volume":"370","author":"Xiang","year":"2020","journal-title":"Science"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"eabh0319","DOI":"10.1126\/sciadv.abh0319","article-title":"Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses","volume":"7","author":"Nambulli","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1038\/s41392-021-00810-1","article-title":"A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants","volume":"6","author":"Yang","year":"2021","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1007\/s00253-017-8644-3","article-title":"Expression of single-domain antibody in different systems","volume":"102","author":"Liu","year":"2018","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"1130","DOI":"10.1016\/j.ijbiomac.2021.10.126","article-title":"A review of plant-based expression systems as a platform for single-domain recombinant antibody production","volume":"193","author":"Malaquias","year":"2021","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Ma, Y., Lee, C.J., and Park, J.S. (2020). Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds. Antibiotics, 9.","DOI":"10.3390\/antibiotics9090541"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.ymben.2024.07.012","article-title":"Scalable, robust, high-throughput expression & purification of nanobodies enabled by 2-stage dynamic control","volume":"85","author":"Hennigan","year":"2024","journal-title":"Metab. Eng."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1007\/978-1-4939-8958-4_17","article-title":"Epitope Mapping via Phage Display from Single-Gene Libraries","volume":"1904","author":"Fuhner","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"108966","DOI":"10.1016\/j.isci.2024.108966","article-title":"A method for rapid nanobody screening with no bias of the library diversity","volume":"27","author":"Tao","year":"2024","journal-title":"iScience"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Jiang, R., Yuan, S., Zhou, Y., Wei, Y., Li, F., Wang, M., Chen, B., and Yu, H. (2024). Strategies to overcome the challenges of low or no expression of heterologous proteins in Escherichia coli. Biotechnol. Adv., 75.","DOI":"10.1016\/j.biotechadv.2024.108417"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1016\/j.bbapap.2008.11.005","article-title":"Outer membrane permeability and antibiotic resistance","volume":"1794","author":"Delcour","year":"2009","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Gawin, A., Ertesv\u00e5g, H., Hansen, S.A.H., Malmo, J., and Brautaset, T. (2020). Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli. BMC Biotech., 20.","DOI":"10.1186\/s12896-020-00615-0"},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Zhang, Z., Tang, R., Zhu, D., Wang, W., Yi, L., and Ma, L. (2017). Non-peptide guided auto-secretion of recombinant proteins by super-folder green fluorescent protein in Escherichia coli. Sci. Rep., 7.","DOI":"10.1038\/s41598-017-07421-3"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1468","DOI":"10.1111\/1751-7915.12819","article-title":"Escherichia coli surface display for the selection of nanobodies","volume":"10","author":"Salema","year":"2017","journal-title":"Microb. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Zhao, S., Yang, G., Xie, X., Yan, G., Wang, F., Chen, W., and Ma, L. (2022). Whole-Cell Display of Phosphotransferase in Escherichia coli for High-Efficiency Extracellular ATP Production. Biomolecules, 12.","DOI":"10.3390\/biom12010139"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"6071","DOI":"10.1007\/s00253-019-09925-6","article-title":"Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner","volume":"103","author":"Liu","year":"2019","journal-title":"Appl. Microbiol. Biotechnol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1654","DOI":"10.1021\/jacsau.4c00129","article-title":"Engineering of Unspecific Peroxygenases Using a Superfolder-Green-Fluorescent-Protein-Mediated Secretion System in Escherichia coli","volume":"4","author":"Yan","year":"2024","journal-title":"JACS Au"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1624","DOI":"10.1002\/bit.27677","article-title":"A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases","volume":"118","author":"Lu","year":"2021","journal-title":"Biotechnol. Bioeng."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Zeng, W., Jia, X., Chi, X., Zhang, X., Li, E., Wu, Y., Liu, Y., Han, J., Ni, K., and Ye, X. (2023). An engineered bispecific nanobody in tetrameric secretory IgA format confers broad neutralization against SARS-CoV-1&2 and most variants. Int. J. Biol. Macromol., 253.","DOI":"10.1016\/j.ijbiomac.2023.126817"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"118521","DOI":"10.1016\/j.ces.2023.118521","article-title":"Multivalent nanobodies with rationally optimized linker and valency for intravitreal VEGF neutralization","volume":"270","author":"Huang","year":"2023","journal-title":"Chem. Eng. Sci."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2308265","DOI":"10.1002\/smll.202308265","article-title":"Bispecific Nanobody-Aptamer Conjugates for Enhanced Cancer Therapy in Solid Tumors","volume":"20","author":"Wang","year":"2024","journal-title":"Small"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"831","DOI":"10.1038\/s41422-022-00700-3","article-title":"Hetero-bivalent nanobodies provide broad-spectrum protection against SARS-CoV-2 variants of concern including Omicron","volume":"32","author":"Ma","year":"2022","journal-title":"Cell Res."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Yu, F., Li, X., Wang, F., Liu, Y., Zhai, C., Li, W., Ma, L., and Chen, W. (2023). TLTC, a T5 exonuclease-mediated low-temperature DNA cloning method. Front. Bioeng. Biotechnol., 11.","DOI":"10.3389\/fbioe.2023.1167534"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"eabe6230","DOI":"10.1126\/science.abe6230","article-title":"Structure-guided multivalent nanobodies block SARS-CoV-2 infection and suppress mutational escape","volume":"371","author":"Koenig","year":"2021","journal-title":"Science"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2072","DOI":"10.1021\/acssuschemeng.1c06647","article-title":"Development of a Hyperthermostable Artificial Scaffold Based on Ultrahigh-Affinity Protein Pairs and Its Application in Cellulose Degradation","volume":"10","author":"Yang","year":"2022","journal-title":"ACS Sustain. Chem. Eng."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"353","DOI":"10.2147\/IJN.S387160","article-title":"A Multivalent and Thermostable Nanobody Neutralizing SARS-CoV-2 Omicron (B.1.1.529)","volume":"18","author":"Lu","year":"2023","journal-title":"Int. J. Nanomed."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"493","DOI":"10.1038\/s41586-024-07487-w","article-title":"Accurate structure prediction of biomolecular interactions with AlphaFold 3","volume":"630","author":"Abramson","year":"2024","journal-title":"Nature"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"846","DOI":"10.1038\/s41594-020-0469-6","article-title":"Neutralizing nanobodies bind SARS-CoV-2 spike RBD and block interaction with ACE2","volume":"27","author":"Huo","year":"2020","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"Lu, Q., Zhang, Z., Li, H., Zhong, K., Zhao, Q., Wang, Z., Wu, Z., Yang, D., Sun, S., and Yang, N. (2021). Development of multivalent nanobodies blocking SARS-CoV-2 infection by targeting RBD of spike protein. J. Nanobiotechnol., 19.","DOI":"10.1186\/s12951-021-00768-w"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"917","DOI":"10.1038\/s41591-021-01318-5","article-title":"Sensitivity of infectious SARS-CoV-2 B.1.1.7 and B.1.351 variants to neutralizing antibodies","volume":"27","author":"Planas","year":"2021","journal-title":"Nat. Med."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1061","DOI":"10.1038\/s41423-021-00648-1","article-title":"Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies","volume":"18","author":"Hu","year":"2021","journal-title":"Cell Mol. Immunol."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Tada, T., Dcosta, B.M., Samanovic, M.I., Herati, R.S., Cornelius, A., Zhou, H., Vaill, A., Kazmierski, W., Mulligan, M.J., and Landau, N.R. (2021). Convalescent-Phase Sera and Vaccine-Elicited Antibodies Largely Maintain Neutralizing Titer against Global SARS-CoV-2 Variant Spikes. mBio, 12.","DOI":"10.1128\/mBio.00696-21"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"8278","DOI":"10.1128\/JVI.03178-13","article-title":"Single-domain antibodies targeting neuraminidase protect against an H5N1 influenza virus challenge","volume":"88","author":"Cardoso","year":"2014","journal-title":"J. Virol."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"6","DOI":"10.1128\/AAC.01802-15","article-title":"Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection","volume":"60","author":"Detalle","year":"2016","journal-title":"Antimicrob. Agents Chemother."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"695","DOI":"10.1146\/annurev-immunol-042617-053327","article-title":"Exploiting Nanobodies\u2019 Singular Traits","volume":"36","author":"Ingram","year":"2018","journal-title":"Annu. Rev. Immunol."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"401","DOI":"10.1146\/annurev-animal-021419-083831","article-title":"Applications of Nanobodies","volume":"9","author":"Muyldermans","year":"2021","journal-title":"Annu. Rev. Anim. 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