Two major autoantigens from Diabetes Mellitus, IA-2 and ZnT8, were expressed as fusion proteins with thioredoxin in Escherichia coli

GUERRA, LUCIANO L.; FACCINETTI, NATALIA I.; ROVITTO, BRUNO D.; IACONO, RUBÉN F.; POSKUS, EDGARDO; TRABUCCHI, ALDANA; VALDEZ, SILVINA N.

Buenos Aires

Congreso; IV LASID Meeting, LXIII Argentinean Immunology Society Meeting, II French-Argentinean Immunology Meeting.; 2015

Two major autoantigens from Diabetes Mellitus, IA-2 and ZnT8, were expressed as fusion proteins with thioredoxin in Escherichia coliGuerra LL, Faccinetti NI, Rovitto BD, Iacono RF, Poskus E, Trabucchi A and Valdez SN.Chair of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), and ?Prof. Ricardo A. Margni? Humoral Immunity Studies Institute (IDEHU), National Research Council (CONICET)-UBAThe aim of this study was to express and recover properly folded recombinant IA-2 and zinc transporter 8 (ZnT8) from Escherichia coli. Sequences coding for the intracellular domain of IA-2 (IA-2ic) and the C-terminal region of ZnT8 were each cloned into pTrxFus vector to obtain them fused to thioredoxin (TrxIA-2ic and TrxZnT8, respectively). GI724 and GI698 E. coli strains were transformed. Both proteins were purified by affinity chromatography from the intracellular soluble fraction. SDS-PAGE and Western Blot were performed using a polyclonal serum to thioredoxin. Proteolytic digestion with trypsin and chymotrypsin were analyzed by Orbitrap mass spectrometry. The immunochemical ability of TrxIA-2ic and TrxZnT8 to compete with [35S]IA-2 or [35S]ZnT8 were assessed by radiometric assays with IA-2A and ZnT8A positive patients sera. Bands compatible with TrxIA-2ic and TrxZnT8 expected theoretical molecular weights (~55.4 and ~37.5 kDa, respectively) were detected from both bacteria strains. TrxIA-2ic expression was achieved in E. coli GI724, yielding 9 mg/L culture, whereas TrxZnT8 purification from strain GI698 yielded 2 mg/L culture. The peptide profile achieved 63.25% and 73.61% coverage for TrxIA-2ic and TrxZnT8 sequences, respectively. Dose-response curves showed similar protein concentration that caused 50% inhibition (53.3-1.1nM for TrxIA-2ic and 41.0-1.4 nM for TrxZnT8). The thirty positive sera evaluated became virtually negative under antigen excess (mean SDs changed from 17.07 to 0.34 for TrxIA-2ic, and 44.88 to 0.78 for TrxZnT8). Recombinant IA-2 and ZnT8 were successfully expressed and purified from E. coli as fusion proteins with thioredoxin. We identify them and demonstrate their proper immunochemical behaviour.