Abstract
BackgroundThe 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).ResultsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0309-2) contains supplementary material, which is available to authorized users.
Highlights
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
The IA-2 structure consists of three domains: the extracellular domain that is separated from the intracellular domain or IA-2ic by a single transmembrane domain
We have previously described the expression of IA-2 in E. coli; the recombinant protein obtained was not stable enough to be implemented in the routine diagnosis of autoimmune Diabetes Mellitus (DM) [50]
Summary
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. IA-2 participates in the cytoplasmic transport of dense core secretory granules (DSG) containing insulin, its subsequent excretion into the extracellular space, biogenesis, storage and recycling, as well as in beta-cell proliferation [8,9,10,11,12]. After exocytosis, calpain mediates the release of IA-2ic from the plasma membrane; a process after which IA-2 travels to the nucleus where its inactive PTP domain binds to the phosphorylated tyrosine present in the STAT5 protein, upregulating gene expression of DSG proteins, including insulin and IA-2 [13,14,15,16]
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