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

GUERRA LL, FACCINETTI NI, TRABUCCHI A, ROVITTO BD, SABLJIC AV, POSKUS E, IACONO RF, VALDEZ SN

BMC BIOTECHNOLOGY

BIOMED CENTRAL LTD

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

1472-6750

Background:Theinsulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of theimmunodominant autoantigens involved in the autoimmune attack to the beta-cellin Type 1 Diabetes Mellitus. In this work we have developed a complete andoriginal process for the production and recovery of the properly foldedintracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic)in Escherichia coli GI698 and GI724 strains. We have alsocarried out the biochemical and immunochemical characterization of TrxIA-2icanddesign variants of non-radiometricimmunoassaysfor the efficient detection of IA-2 autoantibodies (IA-2A).Results:Themain findings can be summarized in the following statements: i) TrxIA-2icexpressionafter 3 h of induction on GI724 strain yielded ¡Ö 10mg of highly pure TrxIA-2ic/Lof culture medium by a single step purification by affinity chromatography, ii)the molecular weight of TrxIA-2ic (55,358Da) could be estimated by SDS-PAGE, size exclusion chromatography and massspectrometry, iii) TrxIA-2ic wasproperly identified by western blot and mass spectrometric analysis ofproteolytic digestions (63.25 % total coverage), iv) excellent immunochemicalbehavior of properly folded full TrxIA-2icwaslegitimized by inhibition or displacement of [35S]IA-2binding from IA-2A presentinArgentinian Type 1 Diabetic patients, v) great stability over time was foundunder proper storage conditions and vi) low cost and environmentally harmlessELISA methods for IA-2A assessment were developed, with colorimetric orchemiluminescent detection.Conclusions:E. coli GI724 strain emerged as a handy sourceof recombinant IA-2ic,achieving high levels of expression as a thioredoxin fusion protein, adequatelyvalidated and applicable to the development of innovative and cost-effectiveimmunoassays for IA-2A detection in most laboratorios.