title: Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application.
abstract: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A).
  
  
  
  The main findings can be summarized in the following statements: i) TrxIA-2icexpression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic(55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2icwas properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2icwas legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection.
  
  
  
  E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Luciano Lucas
authors.initials: LL
authors.lastname: Guerra
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Natalia Inés
authors.initials: NI
authors.lastname: Faccinetti
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Aldana
authors.initials: A
authors.lastname: Trabucchi
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Bruno David
authors.initials: BD
authors.lastname: Rovitto
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Adriana Victoria
authors.initials: AV
authors.lastname: Sabljic
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Edgardo
authors.initials: E
authors.lastname: Poskus
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina.
authors.forename: Ruben Francisco
authors.initials: RF
authors.lastname: Iacono
authors.affiliation: Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina. silval@ffyb.uba.ar.
authors.forename: Silvina Noemí
authors.initials: SN
authors.lastname: Valdez
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D015415:Biomarkers / Q000097:blood; D003922:Diabetes Mellitus, Type 1 / Q000097:blood / Q000175:diagnosis* / Q000276:immunology*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D007159:Immunologic Tests / Q000379:methods; D008297:Male; D008875:Middle Aged; D015202:Protein Engineering / Q000379:methods*; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8 / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013879:Thioredoxins / Q000235:genetics / Q000276:immunology*; D055815:Young Adult
chemical_list: D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D054635:Receptor-Like Protein Tyrosine Phosphatases, Class 8
keywords: Autoantibody; Autoimmunity; Diabetes Mellitus; Escherichia coli; IA-2; Immunoassay; Recombinant protein expression