{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,4]],"date-time":"2026-03-04T09:21:50Z","timestamp":1772616110414,"version":"3.50.1"},"reference-count":40,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2022,4,25]],"date-time":"2022-04-25T00:00:00Z","timestamp":1650844800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Funda\u00e7\u00e3o para a Ci\u00eancia e Tecnologia","doi-asserted-by":"publisher","award":["UIDB\/00709\/2020; UIDP\/00709\/2020; UIDB\/04378\/2020; LA\/P\/0140\/2020; IF\/01459\/2015; SFRH\/BD\/10201\/2020; SFRH\/BD\/147519\/2019; SFRH\/BD\/130068\/2017"],"award-info":[{"award-number":["UIDB\/00709\/2020; UIDP\/00709\/2020; UIDB\/04378\/2020; LA\/P\/0140\/2020; IF\/01459\/2015; SFRH\/BD\/10201\/2020; SFRH\/BD\/147519\/2019; SFRH\/BD\/130068\/2017"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomedicines"],"abstract":"<jats:p>Nucleic acid vaccines have been proven to be a revolutionary technology to induce an efficient, safe and rapid response against pandemics, like the coronavirus disease (COVID-19). Minicircle DNA (mcDNA) is an innovative vector more stable than messenger RNA and more efficient in cell transfection and transgene expression than conventional plasmid DNA. This work describes the construction of a parental plasmid (PP) vector encoding the receptor-binding domain (RBD) of the S protein from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the use of the Design of Experiments (DoE) to optimize PP recombination into mcDNA vector in an orbital shaker. First, the results revealed that host cells should be grown at 42 \u00b0C and the Terrific Broth (TB) medium should be replaced by Luria Broth (LB) medium containing 0.01% L-arabinose for the induction step. The antibiotic concentration, the induction time, and the induction temperature were used as DoE inputs to maximize the % of recombined mcDNA. The quadratic model was statistically significant (p-value &lt; 0.05) and presented a non-significant lack of fit (p-value &gt; 0.05) with a suitable coefficient of determination. The optimal point was validated using 1 h of induction, at 30 \u00b0C, without the presence of antibiotics, obtaining 93.87% of recombined mcDNA. Based on these conditions, the production of mcDNA was then maximized in a mini-bioreactor platform. The most favorable condition obtained in the bioreactor was obtained by applying 60% pO2 in the fermentation step during 5 h and 30% pO2 in the induction step, with 0.01% L-arabinose throughout 5 h. The yield of mcDNA-RBD was increased to a concentration of 1.15 g\/L, when compared to the orbital shaker studies (16.48 mg\/L). These data revealed that the bioreactor application strongly incremented the host biomass yield and simultaneously improved the recombination levels of PP into mcDNA. Altogether, these results contributed to improving mcDNA-RBD biosynthesis to make the scale-up of mcDNA manufacture simpler, cost-effective, and attractive for the biotechnology industry.<\/jats:p>","DOI":"10.3390\/biomedicines10050990","type":"journal-article","created":{"date-parts":[[2022,4,25]],"date-time":"2022-04-25T21:16:28Z","timestamp":1650921388000},"page":"990","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Maximization of the Minicircle DNA Vaccine Production Expressing SARS-CoV-2 RBD"],"prefix":"10.3390","volume":"10","author":[{"given":"Cathy","family":"Ventura","sequence":"first","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"}]},{"given":"Dalinda","family":"Eus\u00e9bio","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"}]},{"given":"Ana M.","family":"Gon\u00e7alves","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0142-5719","authenticated-orcid":false,"given":"Jorge","family":"Barroca-Ferreira","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"}]},{"given":"Diana","family":"Costa","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"}]},{"given":"Zhengrong","family":"Cui","sequence":"additional","affiliation":[{"name":"Division of Molecular Pharmaceutics and Drug Delivery, College of Pharmacy, The University of Texas at Austin, Austin, TX 78712, USA"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6910-7576","authenticated-orcid":false,"given":"Lu\u00eds A.","family":"Passarinha","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"},{"name":"Associate Laboratory i4HB-Institute for Health and Bioeconomy, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2819-516 Caparica, Portugal"},{"name":"UCIBIO-Applied Molecular Biosciences Unit, Departamento de Qu\u00edmica, Faculdade de Ci\u00eancias e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal"},{"name":"Laborat\u00f3rio de F\u00e1rmaco-Toxicologia-UBIMedical, Universidade da Beira Interior, 6200-284 Covilha, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9155-7581","authenticated-orcid":false,"given":"\u00c2ngela","family":"Sousa","sequence":"additional","affiliation":[{"name":"CICS-UBI-Health Science Research Centre, University of Beira Interior, Av. Infante D. Henrique, 6200-506 Covilha, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2022,4,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1016\/j.bj.2021.04.006","article-title":"Role of SARS-CoV-2 and ACE2 variations in COVID-19","volume":"44","author":"Antony","year":"2021","journal-title":"Biomed. J."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Kaneko, S., Takasawa, K., Asada, K., Shinkai, N., Bolatkan, A., Yamada, M., Takahashi, S., Machino, H., Kobayashi, K., and Komatsu, M. (2021). Epigenetic Mechanisms Underlying COVID-19 Pathogenesis. Biomedicines, 9.","DOI":"10.3390\/biomedicines9091142"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"403","DOI":"10.1056\/NEJMoa2035389","article-title":"Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine","volume":"384","author":"Baden","year":"2021","journal-title":"N. Engl. J. 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