IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Expression, purification and immunochemical behaviour of recombinant chimeric thioredoxin proinsulin
Autor/es:
A. TRABUCCHI, L.L. GUERRA, N.I. FACCINETTI, R.F. IACONO, E. POSKUS, S.N VALDEZ
Lugar:
Buzios, Río de Janeiro, Brasil
Reunión:
Congreso; VII Iberoamerican Congress of Biophysics; 2009
Institución organizadora:
Iberoamerican Biophysics Society
Resumen:
It is difficult-to-express native proinsulin in Escherichia coli, due to its relative small size, high proteolytic decay, quaternary structure and its native disulfide pattern. Objective: to express and characterize human proinsulin as a fusion protein with thioredoxin (Trx-PI) in E. coli for its potential use in immunoassays applied to Diabetes Mellitus. Methodology: E. coli GI724 was transformed with p-TrxFus-PI, Trx-PI was obtained from the soluble fraction and it was purified in by affinity chromatography on immobilized phenylarsine oxide. Protein from inclusion bodies was recovered. Competition assays using sera from 30 diabetic patients that scored positive in the standard anti-proinsulin test were carried out. To study the immunochemical behaviour of Trx-PI compared to standard proinsulin (PI), dose-response curves were performed with specific anti-proinsulin polyclonal sera or with a pool of proinsulin antibodies-positive sera. The cross-reactivity between Trx-PI and standard PI was analysed in terms of parallelism of dose-response curves. Results: Trx-PI purified from intracellular soluble fraction yielded 1.5 mg 90-95% pure Trx-PI /L culture. Refolded protein from inclusion bodies was also obtained with high purity. WB analysis showed one band exhibiting the expected mass (21 KDa) for the full-length engineered protein. Competition assays in presence of 1 uM of Trx-PI decreased Standard Deviation Score (sDS) to 1.01 ±1.22 (cutoff value for positivity: sDS= 3.00). Dose-response curves showed parallelism between Trx-PI and standard PI. Conclusions: It was possible to obtain purified human proinsulin as a fusion protein in E. coli. Immunoassays using specific sera, demonstrated full immunoreactivity of Trx-PI indicating integrity of the epitopes involved in the interaction with autoantibodies. Dose-response curves showed a parallel behaviour between Trx-PI and the standard PI, demonstrating that the chimeric molecule retain substantial immunoreactivity associated to the proinsulin moiety.