{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,26]],"date-time":"2026-03-26T18:07:55Z","timestamp":1774548475156,"version":"3.50.1"},"reference-count":34,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2022,12,12]],"date-time":"2022-12-12T00:00:00Z","timestamp":1670803200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"JSPS Grant-in-Aid for Scientific Research","award":["KAKENHI-15H04359"],"award-info":[{"award-number":["KAKENHI-15H04359"]}]},{"name":"JSPS Grant-in-Aid for Scientific Research","award":["18H02385"],"award-info":[{"award-number":["18H02385"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>A large-scale Escherichia coli (E. coli) production of the receptor-binding domain (RBD) of the SARS-CoV-2 could yield a versatile and low-cost antigen for a subunit vaccine. Appropriately folded antigens can potentially elicit the production of neutralizing antisera providing immune protection against the virus. However, E. coli expression using a standard protocol produces RBDs with aberrant disulfide bonds among the RBD\u2019s eight cysteines resulting in the expression of insoluble and non-native RBDs. Here, we evaluate whether E. coli expressing RBD can be used as an antigen candidate for a subunit vaccine. The expressed RBD exhibited native-like structural and biophysical properties as demonstrated by analytical RP-HPLC, circular dichroism, fluorescence, and light scattering. In addition, our E. coli expressed RBD binds to hACE2, the host cell\u2019s receptor, with a binding constant of 7.9 \u00d7 10\u22129 M, as indicated by biolayer interferometry analysis. Our E. coli-produced RBD elicited a high IgG titer in Jcl:ICR mice, and the RBD antisera inhibited viral growth, as demonstrated by a pseudovirus-based neutralization assay. Moreover, the increased antibody level was sustained for over 15 weeks after immunization, and a high percentage of effector and central memory T cells were generated. Overall, these results show that E. coli-expressed RBDs can elicit the production of neutralizing antisera and could potentially serve as an antigen for developing an anti-SARS-CoV-2 subunit vaccine.<\/jats:p>","DOI":"10.3390\/ijms232415744","type":"journal-article","created":{"date-parts":[[2022,12,12]],"date-time":"2022-12-12T04:05:22Z","timestamp":1670817922000},"page":"15744","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["An Escherichia coli Expressed Multi-Disulfide Bonded SARS-CoV-2 RBD Shows Native-like Biophysical Properties and Elicits Neutralizing Antisera in a Mouse Model"],"prefix":"10.3390","volume":"23","author":[{"given":"Subbaian","family":"Brindha","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Takahiro","family":"Yoshizue","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rawiwan","family":"Wongnak","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hitoshi","family":"Takemae","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Epidemiology and Prevention Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-Cho, Fuchu-shi 183-8509, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4187-3217","authenticated-orcid":false,"given":"Mami","family":"Oba","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Epidemiology and Prevention Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-Cho, Fuchu-shi 183-8509, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tetsuya","family":"Mizutani","sequence":"additional","affiliation":[{"name":"Center for Infectious Disease Epidemiology and Prevention Research, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-Cho, Fuchu-shi 183-8509, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3221-4295","authenticated-orcid":false,"given":"Yutaka","family":"Kuroda","sequence":"additional","affiliation":[{"name":"Department of Biotechnology and Life Science, Faculty of Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Nakamachi, Koganei-shi 184-8588, Tokyo, Japan"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Coronaviridae Study Group of the International Committee on Taxonomy of Viruses (2020). The species Severe acute respiratory syndrome-related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2. Nat. Microbiol., 5, 536\u2013544.","DOI":"10.1038\/s41564-020-0695-z"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"4576","DOI":"10.1136\/bmj.m4576","article-title":"COVID-19: What do we know about the late stage vaccine candidates?","volume":"371","author":"Mahase","year":"2020","journal-title":"Brit. Med. J."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"761","DOI":"10.1586\/14760584.2014.912134","article-title":"Current advancements and potential strategies in the development of MERS-CoV vaccines","volume":"13","author":"Zhang","year":"2014","journal-title":"Expert Rev. Vaccines"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Abd El-Lateef, H.M., El-Dabea, T., Khalaf, M.M., and Abu-Dief, A.M. (2022). Development of Metal Complexes for Treatment of Coronaviruses. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23126418"},{"key":"ref_5","first-page":"1","article-title":"Silver nanoparticles (AgNPs) as a metal nano-therapy: Possible mechanisms of antiviral action against COVID-19","volume":"22","year":"2022","journal-title":"Inorg. Nano-Met. Chem."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1007\/s12250-018-0009-2","article-title":"Evaluation of antibody-dependent enhancement of SARS-CoV infection in rhesus macaques immunized with an inactivated SARS-CoV vaccine","volume":"33","author":"Luo","year":"2018","journal-title":"Virol. Sin."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2323","DOI":"10.1002\/1873-3468.14171","article-title":"Single-dose immunisation with a multimerised SARS-CoV-2 receptor binding domain (RBD) induces an enhanced and protective response in mice","volume":"595","author":"Salzer","year":"2021","journal-title":"FEBS Lett."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"165878","DOI":"10.1016\/j.bbadis.2020.165878","article-title":"Insights into SARS-CoV-2 genome, structure, evolution, pathogenesis and therapies: Structural genomics approach","volume":"1866","author":"Naqvi","year":"2020","journal-title":"Biochim. Biophys. Acta Mol. Basis Dis."},{"key":"ref_9","first-page":"55","article-title":"Coronavirus biology and replication: Implications for SARS-CoV-2","volume":"19","author":"Kratzel","year":"2021","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1038\/s41586-020-2180-5","article-title":"Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor","volume":"581","author":"Lan","year":"2020","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1038\/nature02145","article-title":"Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus","volume":"426","author":"Li","year":"2003","journal-title":"Nature"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1016\/j.bbrc.2020.02.071","article-title":"Structure analysis of the receptor binding of 2019-nCoV","volume":"525","author":"Yun","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"613","DOI":"10.1038\/s41423-020-0400-4","article-title":"Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: Implication for development of RBD protein as a viral attachment inhibitor and vaccine","volume":"17","author":"Wanbo","year":"2020","journal-title":"Cell. Mol. Immunol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3309","DOI":"10.1016\/j.jmb.2020.04.009","article-title":"Phylogenetic Analysis and Structural Modeling of SARS-CoV-2 Spike Protein Reveals an Evolutionary Distinct and Proteolytically Sensitive Activation Loop","volume":"432","author":"Jaimes","year":"2020","journal-title":"J. Mol. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"553","DOI":"10.1038\/s41586-021-03594-0","article-title":"Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses","volume":"594","author":"Saunders","year":"2021","journal-title":"Nature"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"21779","DOI":"10.1038\/s41598-020-78711-6","article-title":"Argentinian AntiCovid Consortium Structural and functional comparison of SARS-CoV-2-spike receptor binding domain produced in Pichia pastoris and mammalian cells","volume":"10","author":"Arbeitman","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"P61","DOI":"10.1186\/1475-2859-5-S1-P61","article-title":"Potentials and limitations of prokaryotic and eukaryotic expression systems for recombinant protein production\u2014A comparative view","volume":"5","author":"Bayer","year":"2006","journal-title":"Microb. Cell Fact."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1038\/nbt0412-300a","article-title":"First HEV vaccine approved","volume":"30","author":"Proffitt","year":"2012","journal-title":"Nat. Biotechnol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"145","DOI":"10.1093\/jnci\/djz074","article-title":"Efficacy, safety, and immunogenicity of an Escherichia coli-produced bivalent human papillomavirus vaccine: An interim analysis of a randomized clinical trial","volume":"112","author":"Qiao","year":"2020","journal-title":"J. Natl. Cancer Inst."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1007\/s40265-018-0869-7","article-title":"MenB-FHbp meningococcal group B vaccine (Trumenba): A review in active immunization in individuals aged \u2265 10 years","volume":"78","author":"Shirley","year":"2018","journal-title":"Drugs"},{"key":"ref_21","first-page":"172","article-title":"Recombinant protein expression in Escherichia coli: Advances and challenges","volume":"17","author":"Rosano","year":"2014","journal-title":"Front Microbiol."},{"key":"ref_22","first-page":"1","article-title":"Utilization of Receptor-Binding Domain of SARS-CoV-2 Spike Protein Expressed in Escherichia coli for the Development of Neutralizing Antibody Assay","volume":"14","author":"Tantiwiwat","year":"2022","journal-title":"Mol. Biotechnol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"287","DOI":"10.1016\/j.molimm.2021.12.006","article-title":"Generation and utility of a single-chain fragment variable monoclonal antibody platform against a baculovirus expressed recombinant receptor binding domain of SARS-CoV-2 spike protein","volume":"141","author":"Salem","year":"2021","journal-title":"Mol. Immunol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2013","DOI":"10.1002\/j.1460-2075.1994.tb06471.x","article-title":"The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation","volume":"13","author":"Missiakas","year":"1994","journal-title":"EMBO J."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"56","DOI":"10.1186\/1475-2859-11-56","article-title":"SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm","volume":"11","author":"Lobstein","year":"2012","journal-title":"Microb. Cell Factories"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"574","DOI":"10.1038\/nsmb.1591","article-title":"Converging concepts of protein folding in vitro and in vivo","volume":"16","author":"Hartl","year":"2009","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"630814","DOI":"10.3389\/fmicb.2021.630814","article-title":"Enhanced recombinant protein production under special environmental stress","volume":"12","author":"Chen","year":"2021","journal-title":"Front. Microbiol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1016\/bs.mie.2015.09.031","article-title":"Production of Recombinant Chemokines and Validation of Refolding","volume":"570","author":"Veldkamp","year":"2016","journal-title":"Methods Enzymol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1016\/j.bbrc.2021.03.102","article-title":"EGFR extracellular domain III expressed in Escherichia coli with SEP tag shows improved biophysical and functional properties and generate anti-sera inhibiting cancer cell growth","volume":"555","author":"Brindha","year":"2021","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1703","DOI":"10.3390\/ijms23031703","article-title":"A Multi-Disulfide Receptor-Binding Domain (RBD) of the SARS-CoV-2 Spike Protein Expressed in E. coli Using a SEP-Tag Produces Antisera Interacting with the Mammalian Cell Expressed Spike (S1) Protein","volume":"23","author":"Brindha","year":"2022","journal-title":"Int. J. Mol. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1093\/nar\/gky497","article-title":"BeStSel: A web server for accurate protein secondary structure prediction and fold recognition from the circular dichroism spectra","volume":"46","author":"Micsonai","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"3254","DOI":"10.1021\/bi00793a015","article-title":"Solute perturbation of protein fluorescence. Quenching of the tryptophyl fluorescence of model compounds and of lysozyme by iodide ion","volume":"10","author":"Lehrer","year":"1971","journal-title":"Biochemistry"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2093","DOI":"10.1016\/S0006-3495(01)76183-8","article-title":"Mechanisms of tryptophan fluorescence shifts in proteins","volume":"80","author":"Vivian","year":"2001","journal-title":"Biophys. J."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"383","DOI":"10.1292\/jvms.15-0552","article-title":"Development of a novel detection system for microbes from bovine diarrhea by real-time PCR","volume":"78","author":"Tsuchiaka","year":"2016","journal-title":"J. Vet. Med. Sci."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15744\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T01:39:37Z","timestamp":1760146777000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/24\/15744"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,12,12]]},"references-count":34,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2022,12]]}},"alternative-id":["ijms232415744"],"URL":"https:\/\/doi.org\/10.3390\/ijms232415744","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,12,12]]}}}