IDEHU   05542
INSTITUTO DE ESTUDIOS DE LA INMUNIDAD HUMORAL PROF. RICARDO A. MARGNI
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Improved expression and characterization of human proinsulin fused to thioredoxin in E. coli
Autor/es:
GUERRA, LUCIANO L.; TRABUCCHI, ALDANA; FACCINETTI, NATALIA I.; IACONO, RUBEN F.; POSKUS, EDGARDO; VALDEZ, SILVINA N.
Lugar:
Puerto Madryn, Chubut, Argentina
Reunión:
Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2010
Institución organizadora:
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Resumen:
IMPROVED EXPRESSION AND CHARACTERIZATION OF HUMAN PROINSULIN FUSED TO THIOREDOXIN IN E. COLI Guerra LL, Trabucchi A, Faccinetti NI, Iacono RF, Poskus E, Valdez SN. Chair of Immunology, School of Pharmacy and Biochemistry, UBA and IDEHU, CONICET-UBA E-mail: lguerra@ffyb.uba.ar Native proinsulin (PI) belongs to the class of the difficult-to-express proteins in E.coli, due to its small size, a high proteolytic decay and the necessity to reproduce the native disulfide pattern. Large amounts of properly folded PI are needed to test PI autoantibody of populations at risk for type 1 diabetes.The aim of this study was to recover high yield of properly folded human PI as a fusion to thioredoxin (TrxPI) from inclusion bodies (IB) of E. coli. Methodos: TrxPI was solubilized from IB, refolded in vitro and purified by anion exchange chromatography. It was subjected to mass spectometry analysis and to proteolitic digestion using S. aureus protease V8. To study the immunochemical behaviour of TrxPI compared to standard PI, dose-response curves were performed with specific anti-proinsulin polyclonal sera.Results: TrxPI recovered from IB yielded 10 mg/L culture, and its purity was confirmed by a single peak in RP-HPLC. The product identity and integrity were verified by mass analysis (22,173.5 Da) and mapping with V8. Dose-response curves showed parallelism and identity between TrxPI and standard PI.Conclusions: It was possible to obtain high yield of purified human PI as a fusion protein in E. coli. Proper PI folding was confirmed by biochemical and immunochemical assays indicating integrity of the chimera and the epitopes involved in the interaction with antibodies.