IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Prokariotic expression, purification and immunochemical characterization of Zinc Transporter 8 (ZnT8).
Autor/es:
FACCINETTI, N. I.; GUERRA, L. L.; TRABUCCHI, A.; POSKUS, E.; IACONO, R. F.; VALDEZ, S.N.
Lugar:
Chascomús, Buenos Aires
Reunión:
Congreso; XVI Jornadas Anuales de la Sociedad Argentina de Biología.; 2014
Institución organizadora:
Sociedad Argentina de Biología
Resumen:
ZnT8 is an islet B-cell protein identified as a novel target of humoral autoimmunity in type 1 Diabetes Mellitus. The aim of this study was to express recombinant ZnT8 useful for the development of non-radiometric immunoassays for autoantibodies to ZnT8 (ZnT8A) detection. The C-terminal of ZnT8 was cloned into pTrxFus; E. coli was transformed with pTrxZnT8, cultured at 30°C and induced with Trp. The chimera was purified by affinity or anion exchange chromatography. The intracellular soluble fraction (ISF) and inclusion bodies (IB) were analyzed by SDS-PAGE and Western Blot (WB). Quantitative competition assays with 5 ZnT8A+ patients sera were performed by adding TrxZnT8 (37pM to 0.7uM) to the Radioligand Binding Assay, using [35S]ZnT8 (synthetized with rabbit reticulocyte lysate system). SDS-PAGE and WB showed a band of ~37.5 kDa compatible with TrxZnT8 theoretical mass. Purification of the chimera from ISF (yielding 2mg TrxZnT8/L culture) and IB was achieved. All dose-response curves showed similar protein concentration that caused 50% inhibition (41.0 to 1.4 nM). Recombinant ZnT8 was successfully expressed and purified from E. coli as a fusion protein with Trx. We demonstrate its proper immunochemical behaviour by displacement of [35S]ZnT8 binding to ZnT8A.