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

Microbial Cell Factories

Research

Luciano L. Guerra¹  Silvina N. Valdez¹  Bruno D. Rovitto¹  Ruben F. Iacono¹  Adriana V. Sabljic¹  Edgardo Poskus¹  Silvina S. Bombicino¹  Natalia I. Faccinetti¹  Aldana Trabucchi¹ 

[1] Universidad de Buenos Aires, Facultad de Farmacia y Bioquímica, Departamento de Microbiología, Inmunología, Biotecnología y Genética, Cátedra de Inmunología, Buenos Aires, Argentina;CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral “Prof. Ricardo A. Margni” (IDEHU), Buenos Aires, Argentina;

关键词: Diabetes mellitus;    ZnT8;    Autoantibody;    Recombinant protein expression;    Immunoassays;    Escherichia coli;   

DOI  :  10.1186/s12934-017-0816-4

 received in 2017-07-11, accepted in 2017-11-08,  发布年份 2017

来源: Springer

【 摘 要 】

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.

【 授权许可】

CC BY   
© The Author(s) 2017