Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application

BMC Biotechnology

Research Article

Silvina Noemí Valdez¹  Ruben Francisco Iacono¹  Luciano Lucas Guerra¹  Adriana Victoria Sabljic¹  Edgardo Poskus¹  Natalia Inés Faccinetti¹  Bruno David Rovitto¹  Aldana Trabucchi¹ 

[1] Universidad de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto de Estudios de la Inmunidad Humoral “Prof. Ricardo A. Margni” (IDEHU), Facultad de Farmacia y Bioquímica, Buenos Aires, Argentina;

关键词: IA-2;    Recombinant protein expression;    Diabetes Mellitus;    Autoantibody;    Autoimmunity;    Immunoassay;    Escherichia coli;   

DOI  :  10.1186/s12896-016-0309-2

 received in 2016-06-03, accepted in 2016-10-21,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundThe 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).ResultsThe 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.ConclusionsE. 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.

【 授权许可】

CC BY   
© The Author(s). 2016