{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,27]],"date-time":"2026-03-27T03:17:25Z","timestamp":1774581445413,"version":"3.50.1"},"reference-count":19,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,6,18]],"date-time":"2025-06-18T00:00:00Z","timestamp":1750204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Spinnaker Health Research Foundation"},{"name":"Murdoch University Research Training Programme Scholarship"},{"name":"Perron Institute for Neurological and Translational Science"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MPs"],"abstract":"<jats:p>Autoantibodies targeting cytosolic 5\u2032-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sj\u00f6gren\u2019s syndrome, and systemic lupus erythematosus. While they have diagnostic relevance for IBM, little is known about the autoreactive B cells that produce these antibodies. To address this, we developed a robust protocol for the expression and site-specific biotinylation of recombinant human cN1A in Escherichia coli. The resulting antigen is suitable for generating double-labelled fluorescent baits for the isolation and characterisation of cN1A-specific B cells by flow cytometry. Site-specific biotinylation was achieved using the AviTag and BirA ligase, preserving the protein\u2019s structure and immunoreactivity. Western blot analysis confirmed that the biotinylated cN1A was recognised by both human and rabbit anti-cN1A antibodies. Compared to conventional chemical biotinylation, this strategy minimises structural alterations that may affect antigen recognition. This approach provides a reliable method for producing biotinylated antigens for use in immunological assays. While demonstrated here for cN1A, the protocol can be adapted for other autoantigens to support studies of antigen-specific B cells in autoimmune diseases.<\/jats:p>","DOI":"10.3390\/mps8030066","type":"journal-article","created":{"date-parts":[[2025,6,19]],"date-time":"2025-06-19T06:11:07Z","timestamp":1750313467000},"page":"66","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Expression and Site-Specific Biotinylation of Human Cytosolic 5\u2032-Nucleotidase 1A in Escherichia coli"],"prefix":"10.3390","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9979-6074","authenticated-orcid":false,"given":"Nataliya","family":"Slater","sequence":"first","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2576-3309","authenticated-orcid":false,"given":"Anuradha","family":"Sooda","sequence":"additional","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frank L.","family":"Mastaglia","sequence":"additional","affiliation":[{"name":"Perron Institute for Neurological and Translational Science, University of Western Australia, Nedlands, WA 6009, Australia"},{"name":"Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Crawley, WA 6009, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8632-641X","authenticated-orcid":false,"given":"Sue","family":"Fletcher","sequence":"additional","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6438-9225","authenticated-orcid":false,"given":"Mark","family":"Watson","sequence":"additional","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6591-1444","authenticated-orcid":false,"given":"Merrilee","family":"Needham","sequence":"additional","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"},{"name":"Perron Institute for Neurological and Translational Science, University of Western Australia, Nedlands, WA 6009, Australia"},{"name":"School of Medicine, University of Notre Dame Australia, Fremantle, WA 6160, Australia"},{"name":"Department of Neurology, Fiona Stanley Hospital, Murdoch, WA 6150, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6081-8494","authenticated-orcid":false,"given":"Jerome D.","family":"Coudert","sequence":"additional","affiliation":[{"name":"Personalised Medicine Center, Murdoch University, Murdoch, WA 6150, Australia"},{"name":"Perron Institute for Neurological and Translational Science, University of Western Australia, Nedlands, WA 6009, Australia"},{"name":"Centre for Neuromuscular & Neurological Disorders, University of Western Australia, Crawley, WA 6009, Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,6,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"509","DOI":"10.1038\/s41581-023-00720-1","article-title":"Pathogenesis of autoimmune disease","volume":"19","author":"Pisetsky","year":"2023","journal-title":"Nat. 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