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

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.

Expression of recombinant glutamic acid decarboxylase in insect larvae and its application in an immunoassay for the diagnosis of autoimmune diabetes mellitus

“…As previously described in our earlier work 28 , with some modifications, the tracer [ 35 S]GAD65 was obtained by in vitro transcription/translation of cDNA encoding the human GAD65 using a rabbit reticulocyte lysate system (Promega, Madison, WI, USA) in the presence of [ 35 S]-methionine (New England, Nuclear, Boston, MA, USA), according to the manufacturer’s instructions. Translation products were diluted in RBA buffer (0.02 M Tris–HCl, 0.15 M NaCl, pH 7.4, 0.1% v/v Tween 20) and applied to a PD10 column (Pharmacia-LKB Biotechnology, Uppsala, Sweden) in order to remove free [ 35 S]-methionine.…”

“…Quantitative competition assays were performed by a standard radioimmunoassay (RIA), as previously described in our earlier work 28 . The RIA was carried out by incubating 2.5 µL of 5 GADA-positive type 1 diabetic patient sera with 10,000 cpm of [ 35 S]GAD65 in the presence of serial concentrations (0.62 nM-1.4 µM) of purified R .…”

“…The same as described in Guerra et al . 28 , the immunochemical identities and parallelism between inhibitory dose-response curves obtained with Sf 9-GAD65, R . nu -GAD65 and S .…”

Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus

“…TrxZnT8 from the ISF was purified by means of affinity chromatography following the protocol previously described [ 31 , 33 ]. Briefly, the resin was based on an agarosa support covalently modified with phenylarsine oxide, which permitted the binding of proteins containing vicinal dithiol residues, as it occurs in Trx [ 34 ].…”

Inmunoensayo Multiplex Para El Diagnóstico Simultáneo De Diabetes Mellitus Autoinmune Y Enfermedad Celíaca

“…Las proteínas recombinantes GAD e IA-2ic se expresaron en E. coli como proteínas de fusión con tiorredoxina (TrxGAD y TrxIA-2ic, respectivamente) y se purificaron por cromatografía de afinidad siguiendo el protocolo previamente descripto 11,12 . La resina se basó en un soporte de agarosa modificado covalentemente con óxido de fenilarsina que permitió la unión de proteínas que contenían residuos de ditiol vecinos, tal como la Trx 13 .…”