Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

BMC Biotechnol. 2016 Nov 24;16(1):84. doi: 10.1186/s12896-016-0309-2.


Background: 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).

Results: The main findings can be summarized in the following statements: i) TrxIA-2ic expression 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-2ic was 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-2ic was 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.

Conclusions: 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.

Keywords: Autoantibody; Autoimmunity; Diabetes Mellitus; Escherichia coli; IA-2; Immunoassay; Recombinant protein expression.

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Biomarkers / blood
  • Diabetes Mellitus, Type 1 / blood
  • Diabetes Mellitus, Type 1 / diagnosis*
  • Diabetes Mellitus, Type 1 / immunology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Female
  • Humans
  • Immunologic Tests / methods
  • Male
  • Middle Aged
  • Protein Engineering / methods*
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8 / genetics
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8 / immunology*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / immunology
  • Thioredoxins / genetics
  • Thioredoxins / immunology*
  • Young Adult


  • Biomarkers
  • Recombinant Fusion Proteins
  • Thioredoxins
  • Receptor-Like Protein Tyrosine Phosphatases, Class 8