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.
Highlights
In the present work we described the recombinant production and characterization of heterodimeric construction Zinc transporter 8 (ZnT8)-Arg-Trp325 fused to thioredoxin using a high-performance expression system such as Escherichia coli
Among the major autoantibodies employed in the diagnosis of autoimmune diabetes mellitus (DM), autoantibodies to ZnT8 (ZnT8A) is the most recently described humoral marker, complementing those already used such as insulin/proinsulin autoantibodies (IAA/PAA), glutamic acid decarboxylase autoantibodies (GADA), and insulinoma associated protein tyrosine phosphatase 2 autoantibodies (IA-2A)
Western blot (WB) analysis of intracellular soluble fraction (ISF) and inclusion bodies (IB) with polyclonal serum to Trx showed one band with the expected molecular weight (MW, ≈ 36.8 kDa) for the full-length engineered protein, whereas such bands were absent in the ISF and IB from non-transformed bacteria under the same experimental conditions (Additional file 1: Figure S1)
Summary
In 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. Zinc transporter 8 (ZnT8) is a multipass transmembrane protein which has been identified as a novel autoantigen in patients with diabetes mellitus (DM), such as in type. Among the major autoantibodies employed in the diagnosis of autoimmune DM, autoantibodies to ZnT8 (ZnT8A) is the most recently described humoral marker, complementing those already used such as insulin/proinsulin autoantibodies (IAA/PAA), glutamic acid decarboxylase autoantibodies (GADA), and insulinoma associated protein tyrosine phosphatase 2 autoantibodies (IA-2A). ZnT8A constitutes an additional prevalent marker to the preexisting triad, enabling the appropriate classification of diabetic patients as autoimmune DM and improving the efficacy of treatment
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