Expression and Site-Specific Biotinylation of Human Cytosolic 5'-Nucleotidase 1A in <i>Escherichia coli</i>.

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 confi

SUBMITTER: Slater N 

PROVIDER: S-EPMC12195718 | biostudies-literature | 2025 Jun

REPOSITORIES: biostudies-literature