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

FACCINETTI, NATALIA I.; GUERRA, LUCIANO L.; SABLJIC, ADRIANA V.; BOMBICINO, SILVINA S.; ROVITTO, BRUNO D.; IACONO, RUBEN F.; POSKUS, EDGARDO; TRABUCCHI, ALDANA; VALDEZ, SILVINA N.

MICROBIAL CELL FACTORIES

BIOMED CENTRAL LTD

Año: 2017 vol. 16

1475-2859

Background: 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 Escherichiacoli. In addition, we apply this novel recombinant antigen in a non-radiometric method, with high sensitivity, low operational complexity and lower costs.Results: 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 TrxZnT8to 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 deviationscores (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 assayshowed 51.7% of sensitivity and 97.1% of specificity.Conclusions: 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.