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Journal article
Expression and Site-Specific Biotinylation of Human Cytosolic 5′-Nucleotidase 1A in Escherichia coli
Methods and protocols, Vol.8(3), 66
2025
PMID: 40559454
Abstract
Autoantibodies targeting cytosolic 5′-nucleotidase 1A (cN1A) are found in several autoimmune diseases, including inclusion body myositis (IBM), Sjögren’s 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’s 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.
Details
- Title
- Expression and Site-Specific Biotinylation of Human Cytosolic 5′-Nucleotidase 1A in Escherichia coli
- Authors/Creators
- Nataliya Slater - Murdoch UniversityAnuradha Sooda - Murdoch UniversityFrank L. Mastaglia - Perron Institute for Neurological and Translational ScienceSue Fletcher - Murdoch UniversityMark Watson - Murdoch UniversityMerrilee Needham - Murdoch UniversityJerome D. Coudert - Murdoch University
- Publication Details
- Methods and protocols, Vol.8(3), 66
- Publisher
- MDPI
- Number of pages
- 21
- Grant note
- Perron Institute for Neurological and Translational Science Murdoch University Research Training Programme Scholarship Spinnaker Health Research Foundation
- Identifiers
- 991005787887907891
- Copyright
- © 2025 by the authors.
- Murdoch Affiliation
- Health Futures Institute; Personalised Medicine Centre; Genomics Core Research Facility
- Language
- English
- Resource Type
- Journal article
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