BACTERIAL OVEREXPRESSION, PURIFICATION, AND RECONSTITUTION OF THE CARNITINE ACYLCARNITINE CARRIER FROM RAT-LIVER MITOCHONDRIA/

The carnitine/acylcarnitine carrier from rat liver mitochondria was ov erexpressed in Escherichia coli. The expressed protein, recovered as i nclusion bodies, was solubilized with sarkosyl and purified by Sephade x G-200 and celite chromatography. A yield of 15 mg of purified transp ort protein per liter of cell culture was obtained. Upon reconstitutio n into liposomes, the purified carrier catalyzed a [H-3]carnitine/carn itine exchange inhibited by maleimides, mercurials, and sulfobetaines. Carnitine esters of various lengths were also transported. The K-m fo r carnitine uptake was 0.47 +/- 0.11 mM, the V-max of the exchange was 0.78 +/- 0.24 mmol/min per gram of protein, and the Ki for octanoylca rnitine was 13.5 +/- 4.3 mu M. The transport properties of the recombi nant carrier were virtually identical to those of the native transport er. These studies represent the first overexpression of the functional ly active mitochondrial carnitine/acylcarnitine carrier, thus enabling structure/function analysis of this protein by site-directed mutagene sis. (C) 1998 Academic Press.