PMID- 29132366 OWN - NLM STAT- MEDLINE DCOM- 20180712 LR - 20181113 IS - 1475-2859 (Electronic) IS - 1475-2859 (Linking) VI - 16 IP - 1 DP - 2017 Nov 13 TI - Prokaryotic expression and characterization of the heterodimeric construction of ZnT8 and its application for autoantibodies detection in diabetes mellitus. PG - 196 LID - 10.1186/s12934-017-0816-4 [doi] LID - 196 AB - 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 ( approximately 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 muM 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 microM), 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. 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. FAU - Faccinetti, Natalia I AU - Faccinetti NI AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Guerra, Luciano L AU - Guerra LL AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Sabljic, Adriana V AU - Sabljic AV AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Bombicino, Silvina S AU - Bombicino SS AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Rovitto, Bruno D AU - Rovitto BD AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Iacono, Ruben F AU - Iacono RF AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Poskus, Edgardo AU - Poskus E AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Trabucchi, Aldana AU - Trabucchi A AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. FAU - Valdez, Silvina N AU - Valdez SN AD - Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Departamento de Microbiologia, Inmunologia, Biotecnologia y Genetica, Catedra de Inmunologia, Buenos Aires, Argentina. silval@ffyb.uba.ar. AD - CONICET-Universidad de Buenos Aires, Instituto de Estudios de la Inmunidad Humoral "Prof. Ricardo A. Margni" (IDEHU), Buenos Aires, Argentina. silval@ffyb.uba.ar. LA - eng GR - PICT 2014 1928/FONCYT Program of the National Agency for Science and Technology Promotion (ANPCYT)/ PT - Journal Article DEP - 20171113 PL - England TA - Microb Cell Fact JT - Microbial cell factories JID - 101139812 RN - 0 (Antigens) RN - 0 (Autoantibodies) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Zinc Transporter 8) RN - 52500-60-4 (Thioredoxins) SB - IM MH - Animals MH - Antigens/genetics/immunology MH - Autoantibodies/*blood/immunology MH - Diabetes Mellitus, Type 1/*immunology MH - Enzyme-Linked Immunosorbent Assay/economics/methods MH - Escherichia coli/*genetics MH - Humans MH - Rabbits MH - Recombinant Fusion Proteins/chemistry/genetics/immunology/isolation & purification MH - Thioredoxins/chemistry/genetics MH - Zinc Transporter 8/*genetics/*immunology PMC - PMC5683521 OTO - NOTNLM OT - Autoantibody OT - Diabetes mellitus OT - Escherichia coli OT - Immunoassays OT - Recombinant protein expression OT - ZnT8 EDAT- 2017/11/15 06:00 MHDA- 2018/07/13 06:00 PMCR- 2017/11/13 CRDT- 2017/11/15 06:00 PHST- 2017/07/11 00:00 [received] PHST- 2017/11/08 00:00 [accepted] PHST- 2017/11/15 06:00 [entrez] PHST- 2017/11/15 06:00 [pubmed] PHST- 2018/07/13 06:00 [medline] PHST- 2017/11/13 00:00 [pmc-release] AID - 10.1186/s12934-017-0816-4 [pii] AID - 816 [pii] AID - 10.1186/s12934-017-0816-4 [doi] PST - epublish SO - Microb Cell Fact. 2017 Nov 13;16(1):196. doi: 10.1186/s12934-017-0816-4.