HIGH-LEVEL EXPRESSION OF FUNCTIONAL-RAT NEURONAL NITRIC-OXIDE SYNTHASE IN ESCHERICHIA-COLI

The neuronal nitric oxide synthase (nNOS) has been successfully overex pressed in Escherichia coli, with average yields of 125-150 nmol (20-2 4 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned in to the pCW vector under the control of the tac promotor and was coexpr essed with the chaperonins groEL and groES in the protease-deficient B L21 strain off. coli. The enzyme produced is replete with heme and fla vins and, after overnight incubation with tetrahydrobiopterin, contain s 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated a s a predominantly high-spin heme protein and demonstrates spectral pro perties that are identical to those of nNOS isolated from stably trans fected human kidney 293 cells, It binds N-omega-nitroarginine dependen t on the presence of bound tetrahydrobiopterin and exhibits a K-d Of 4 5 nM. The enzyme is completely functional; the specific activity is 45 0 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS fo r use in mechanistic and structure/function studies, as well as for dr ug design and development.