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

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-2(ic)) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2(ic)and 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-2(ic) expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2(ic)/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2(ic) (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2(ic) 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-2(ic) was legitimized by inhibition or displacement of [(35)S]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-2(ic), 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12896-016-0309-2) contains supplementary material, which is available to authorized users.