RAPISARDA Viviana Andrea
congresos y reuniones científicas
Purification and partial characterization of a cytosolic NADH dehydrogenase-2 from E. coli
VILLEGAS, J. M.; VOLENTINI, S. I.; RINTOUL, M. R.; RAPISARDA, V. A.
San Miguel de Tucuman, Tucuman, Argentina
Congreso; XLV Annual Meeting of SAIB; 2009
Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a membrane-bound flavoprotein with cupric-reductase activity. Preliminary studies suggested that the C-terminal 43 aminoacids region is involved in the enzyme anchorage to the membrane. The aim of this work was to obtain a water soluble protein that allows a detergent-free purification. To achieve this goal, we generated a NDH-2 truncated version, Trun-3, where the mentioned C-terminal fragment was deleted. The mutant gene was cloned in frame into the expression vector pETG-30A immediately downstream from 6xHis residues. By ultracentrifugation and SDS-PAGE, we localized Trun-3 in the cytosolic fraction. Afterwards, it was purified by inmobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. The purified protein showed NADH:duroquinone, NADH:MTT and NADH:Cu(II) oxidorreductase activities, always in the presence of 80 ìM FAD. Spectroscopic measurements suggest the absence of FAD bound to the enzyme. The purified water soluble version of NDH-2 will be useful for further characterization of its functional domains.