INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Structural and biochemical studies of a recombinant cytochrome P450 reductase (rTcCPR-A) in Trypanosoma cruzi. Analysis of a coexpression system with chaperones.
Autor/es:
SCHLESINGER, M.; PORTAL, P.; FERNÁNDEZ VILLAMIL, S.; ALONSO, G.; TORRES, H.N.; FLAWIÁ, M.M.; PAVETO, C.
Lugar:
Rosario, Santa Fe, Argentina
Reunión:
Congreso; VIII Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2008
Institución organizadora:
Sociedad Argentina de Protozzología
Resumen:
Recently, three different genes codifying for the cytochrome P450 reductases TcCPR-A, TcCPR-B and TcCPR-C have been identified in our laboratory in Trypanosoma cruzi. The aminoacidic sequences present the characteristic binding domains to FMN, FAD and NADPH, and have 11% of identity, differing mainly in their amino-end. The three enzymes have been cloned and expressed in bacteria and an activity assay was performed by reducing the cytochrome c in a totally NADPH-dependent manner, being the activity partially inhibited by DPI (a flavoprotein inhibitor). With the aim of identifying the TcCPR-A role in the redox metabolism of the parasite, we continued with the biochemical and structural characterization. In order to produce the soluble protein with a high degree of purity and yield we coexpressed it along with chaperons. We obtained the best result with the cotransformation of the pKJE7 vector, which expresses the dnaK-dnaJ-grpE chaperons, and the pET-TcCPR-A plasmid. To show the cofactor dependence we carried out activity assays with the purified protein obtained using Ni-NTA agarosa columns and dialyzed to eliminate the contaminating free flavins. The absorption spectrum within the visible region showed the characteristic picks of flavoproteins and the extinction coefficient at 280 nm was 75830 M-1 cm-1. Nowadays we are studying the presence of FAD and FMN in the purified recombinant protein with the HPLC technique.