Characterization of Rat Neuronal Nitric Oxide Synthase Expressed in Saccharomyces cerevisiae

Abstract

A cDNA encoding rat neuronal nitric oxide synthase (nNOS) was cloned into the yeast expression vector pMA56 to generate pA379. Transformation of Saccharomyces cerevisiae strain BJ2168 with this plasmid resulted in the synthesis of nNOS at levels of 0.5–1.0 mg/liter. The protein is localized in the cytosol and is catalytically active as determined by the conversion of [³H]-l-arginine to [³H]-l-citrulline and NO. The enzyme was purified by calmodulin–Sepharose affinity chromatography and its catalytic activity was found to be both calcium and calmodulin dependent. Overexpression of nNOS in S. cerevisiae and purification of the recombinant protein will facilitate detailed characterization of this important enzyme.