dc.creator | Guerra, Luciano Lucas | |
dc.creator | Faccinetti, Natalia Ines | |
dc.creator | Trabucchi, Aldana | |
dc.creator | Rovitto, Bruno David | |
dc.creator | Sabljic, Adriana Victoria | |
dc.creator | Poskus, Edgardo | |
dc.creator | Iacono, Ruben Francisco | |
dc.creator | Valdez, Silvina Noemi | |
dc.date.accessioned | 2018-06-06T20:04:43Z | |
dc.date.accessioned | 2018-11-06T11:39:57Z | |
dc.date.available | 2018-06-06T20:04:43Z | |
dc.date.available | 2018-11-06T11:39:57Z | |
dc.date.created | 2018-06-06T20:04:43Z | |
dc.date.issued | 2016-11 | |
dc.identifier | Guerra, Luciano Lucas; Faccinetti, Natalia Ines; Trabucchi, Aldana; Rovitto, Bruno David; Sabljic, Adriana Victoria; et al.; Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application; BioMed Central; Bmc Biotechnology; 16; 1; 11-2016; 1-19 | |
dc.identifier | 1472-6750 | |
dc.identifier | http://hdl.handle.net/11336/47586 | |
dc.identifier | CONICET Digital | |
dc.identifier | CONICET | |
dc.identifier.uri | http://repositorioslatinoamericanos.uchile.cl/handle/2250/1857208 | |
dc.description.abstract | Background
The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A).
Results
The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection.
Conclusions
E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories. | |
dc.language | eng | |
dc.publisher | BioMed Central | |
dc.relation | info:eu-repo/semantics/altIdentifier/url/http://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-016-0309-2 | |
dc.relation | info:eu-repo/semantics/altIdentifier/doi/https://doi.org/10.1186/s12896-016-0309-2 | |
dc.rights | https://creativecommons.org/licenses/by-nc-sa/2.5/ar/ | |
dc.rights | info:eu-repo/semantics/openAccess | |
dc.subject | RECOMBINANT PROTEIN EXPRESSION | |
dc.subject | DIABETES MELLITUS | |
dc.subject | AUTOANTIBODY | |
dc.subject | AUTOIMMUNITY | |
dc.subject | IMMUNOASSAY | |
dc.subject | ESCHERICHIA COLI | |
dc.title | Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application | |
dc.type | Artículos de revistas | |
dc.type | Artículos de revistas | |
dc.type | Artículos de revistas | |