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Here we report the scalable expression and purification of soluble, immunologically active, SARS-CoV-2 Ncap in Escherichia coli. Codon-optimised synthetic genes encoding the original Ncap sequence and four common variants with an N-terminal 6His affinity tag (sequence MHHHHHHG) were cloned into an inducible expression vector carrying a regulated bacteriophage T5 synthetic promoter controlled by lac operator binding sites. The constructs were used to express Ncap proteins and protocols developed which allow efficient production of purified Ncap with yields of over 200\u2005mg per litre of culture media. These proteins were deployed in ELISA assays to allow comparison of their responses to human sera. Our results suggest that there was no detectable difference between the 6His-tagged and untagged original Ncap proteins but there may be a slight loss of sensitivity of sera to other Ncap isolates.<\/jats:p>","DOI":"10.1042\/bcj20240019","type":"journal-article","created":{"date-parts":[[2024,5,7]],"date-time":"2024-05-07T13:28:14Z","timestamp":1715088494000},"page":"669-682","update-policy":"https:\/\/doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in <i>Escherichia coli<\/i>"],"prefix":"10.1042","volume":"481","author":[{"given":"Emma L.","family":"Brudenell","sequence":"first","affiliation":[{"name":"1Sheffield Institute for Nucleic Acids and Florey Institute, Section of Infection and Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 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Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hailey R.","family":"Hornsby","sequence":"additional","affiliation":[{"name":"1Sheffield Institute for Nucleic Acids and Florey Institute, Section of Infection and Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"John F.","family":"Darby","sequence":"additional","affiliation":[{"name":"1Sheffield Institute for Nucleic Acids and Florey Institute, Section of Infection and Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Junxiao","family":"Dai","sequence":"additional","affiliation":[{"name":"2Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ellen","family":"Liggett","sequence":"additional","affiliation":[{"name":"2Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kathleen M.","family":"Cain","sequence":"additional","affiliation":[{"name":"2Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Perdita E.","family":"Barran","sequence":"additional","affiliation":[{"name":"2Michael Barber Centre for Collaborative Mass Spectrometry, Department of Chemistry, Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1 7DN, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thushan I.","family":"de Silva","sequence":"additional","affiliation":[{"name":"1Sheffield Institute for Nucleic Acids and Florey Institute, Section of Infection and Immunity, Division of Clinical Medicine, School of Medicine and Population Health, The University of Sheffield, Beech Hill Road, Sheffield S10 2RX, U.K."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5082-1443","authenticated-orcid":false,"given":"Jon R.","family":"Sayers","sequence":"additional","affiliation":[{"name":"1Sheffield Institute for Nucleic 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