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

GUERRA, LUCIANO L.; ROVITTO, BRUNO D.; IACONO, RUBEN F.; FACCINETTI, NATALIA I.; SABLJIC, ADRIANA V.; VALDEZ, SILVINA N.; TRABUCCHI,ALDANA; POSKUS, EDGARDO; GUERRA, LUCIANO L.; ROVITTO, BRUNO D.; FACCINETTI, NATALIA I.; IACONO, RUBEN F.; SABLJIC, ADRIANA V.; VALDEZ, SILVINA N.; TRABUCCHI,ALDANA; POSKUS, EDGARDO

BMC BIOTECHNOLOGY

BIOMED CENTRAL LTD

Lugar: Londres; Año: 2016 vol. 16 p. 1 - 19

1472-6750

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 purificationby 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 massspectrometric 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 colorimetricor 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 innovativeand cost-effective immunoassays for IA-2A detection in most laboratories.