Characterization of Rat Neuronal Nitric Oxide Synthase Expressed inSaccharomyces cerevisiae

摘要

ABSTRACTA cDNA encoding rat neuronal nitric oxide synthase (nNOS) was cloned into the yeast expression vector pMA56 to generate pA379. Transformation ofSaccharomyces cerevisiaestrain 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 inS. cerevisiaeand purification of the recombinant protein will facilitate detailed characterization of this important enz