Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus.
Microb Cell Fact
; 16(1): 196, 2017 Nov 13.
Article
em En
| MEDLINE
| ID: mdl-29132366
ABSTRACT
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 Escherichia coli. 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 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.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.Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Autoanticorpos
/
Diabetes Mellitus Tipo 1
/
Escherichia coli
/
Transportador 8 de Zinco
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
Microb Cell Fact
Assunto da revista:
BIOTECNOLOGIA
/
MICROBIOLOGIA
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Argentina