Expression of the autoantigen Insulinoma-Associated Tyrosine Phosphatase 2 (IA-2) fused to thioredoxin in E. coli.

GUERRA, L. L.; FACCINETTI, N. I.; IACONO, R. F.; POSKUS, E.; TRABUCCHI, A.; VALDEZ, S.N.

Chascomús, Buenos Aires

Congreso; XVI Jornadas Anuales de la Sociedad Argentina de Biología.; 2014

Sociedad Argentina de Biología

IA-2, a transmembrane protein from pancreatric β cells, is an antigen in autoimmune diabetes and autoantibodies to it are early markers of the disease. The aim of this study was to express and recover properly folded IA-2 from Escherichia coli. The sequence coding for the intracellular domain of IA-2 (IA-2ic) was cloned into pTrxFus vector to obtain it fused to thioredoxin (TrxIA-2ic). E. coli GI724 and GI698 were transformed with pTrxIA-2ic, cultured at 30°C and protein expression was induced with Trp 100 µg/mL at 37°C or 20°C, respectively. After induction at different times, cells lysates were obtained and TrxIA-2ic was purified by affinity chromatography from the intracellular soluble fraction. SDS-PAGE and Western Blot analysis revealed a band compatible with TrxIA-2ic expected molecular weight (~55.4 kDa) from both bacteria strains. Higher expression was achieved in E. coli GI724, yielding 0.72 mg of >99% pure TrxIA-2ic/L culture. The immunochemical behaviour of TrxIA-2ic was assessed qualitatively by incubating 30 IA-2A(+) patients sera with [35S]IA-2 (obtained by rabbit reticulocyte lysate system) in the presence of 0.2 μM TrxIA-2ic. All sera became virtually negative under antigen excess (mean Standard Deviation scores changed from 17.07 to 0.34). It was possible to obtain purified IA-2 in E. coli as a fusion protein. The integrity of epitopes involved in the interaction with antibodies was confirmed.