Title |
Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus
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Published in |
Microbial Cell Factories, November 2017
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DOI | 10.1186/s12934-017-0816-4 |
Pubmed ID | |
Authors |
Natalia I. Faccinetti, Luciano L. Guerra, Adriana V. Sabljic, Silvina S. Bombicino, Bruno D. Rovitto, Ruben F. Iacono, Edgardo Poskus, Aldana Trabucchi, Silvina N. Valdez |
Abstract |
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. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs. ZnT8 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 [(35)S]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 [(35)S]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. It 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. |
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