PURIFICATION AND PARTIAL CHARACTERIZATION OF A CYTOSOLIC MUTANT NADH DEHYDROGENASE-2 FROM E. coli

VILLEGAS J. M.; VOLENTINI, S. I.; RINTOUL M. R.; RAPISARDA, V. A.

S M de Tucuman

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en; 2009

SAIB

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 were able to localize trun-3 in the cytosolic fraction. Afterwards, the mutant version 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 mM FAD. Spectroscopic measurements suggest the absence of FAD bound to the enzyme. By the generation of a water soluble protein, we were able to easily obtain a purified FAD dependent active version of NDH-2 that could be useful for further characterization of its functional domains.