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

Abstract

BackgroundIn the present work we described the recombinant production and characterization of heterodimeric construction ZnT8-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs.ResultsZnT8 was expressed in E. coli as a fusion protein with thioredoxin (TrxZnT8). After 3 h for induction, recombinant protein was obtained from the intracellular soluble fraction and from inclusion bodies and purified by affinity chromatography. The expression and purification steps, analyzed by SDS-PAGE and western blot, revealed a band compatible with TrxZnT8 expected theoretical molecular weight (≈ 36.8 kDa). The immunochemical ability of TrxZnT8 to compete with [35S]ZnT8 (synthesized with rabbit reticulocyte lysate system) was assessed qualitatively by incubating ZnT8A positive patient sera in the presence of 0.2–0.3 μM TrxZnT8. Results were expressed as standard deviation scores (SDs). All sera became virtually negative under antigen excess (19.26–1.29 for TrxZnT8). Also, radiometric quantitative competition assays with ZnT8A positive patient sera were performed by adding TrxZnT8 (37.0 pM–2.2 µM), using [35S]ZnT8. All dose–response curves showed similar protein concentration that caused 50% inhibition (14.9–0.15 nM for TrxZnT8). On the other hand, preincubated bridge ELISA for ZnT8A detection was developed. This assay showed 51.7% of sensitivity and 97.1% of specificity.ConclusionsIt was possible to obtain with high-yield purified heterodimeric construction of ZnT8 in E. coli and it was applied in cost-effective immunoassay for ZnT8A detection.Electronic supplementary materialThe online version of this article (10.1186/s12934-017-0816-4) contains supplementary material, which is available to authorized users.

Strategies for Successful Over-Expression of Human Membrane Transport Systems Using Bacterial Hosts: Future Perspectives

“…The codon-optimized sequence coding for the C-terminal domain of the ZnT8 transporter was cloned in a specific plasmid and the corresponding peptide was produced as thioredoxin (Trx), tagged either in inclusion bodies using the GI724 strain or in the soluble fraction of cell lysate culturing GI698 cells ( Table 3 ). The peptide was successfully purified in a properly folded state that could be useful for developing new low-cost tests for the detection of ZnT8 autoantibodies [ 45 ].…”

Novel Flow Cytometric Immunoassay for Detection of Proinsulin Autoantibodies in Diabetes Mellitus Employing a Recombinant Autoantigen Expressed in E. coli

“…On the other hand, TrxPI was isolated from the IB by a first step of solubilization with 8 M urea in 0.1 M Tris, pH 8.5 followed by an oxidative refolding process through dialysis at 4°C against 0.5 M L-arginine, 50 mM Tris-HCl, 5 mM EDTA, 5 mM reduced glutathione and 0.5 mM oxidized glutathione, pH 9.5 (24). The refolded protein´s buffer was exchanged to PBS using a PD-10 desalting column and the protein was stored in fractions with 50 mM 2ME and 0.1% w/v aprotinin.…”