High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.

Abstract

The neuronal nitric oxide synthase (nNOS) has been successfully overexpressed in Escherichia coli, with average yields of 125-150 nmol (20-24 mg) of enzyme per liter of cells. The cDNA for nNOS was subcloned into the pCW vector under the control of the tac promotor and was coexpressed with the chaperonins groEL and groES in the protease-deficient BL21 strain ofE. coli. The enzyme produced is replete with heme and flavins and, after overnight incubation with tetrahydrobiopterin, contains 0.7 pmol of tetrahydrobiopterin per pmol of nNOS. nNOS is isolated as a predominantly high-spin heme protein and demonstrates spectral properties that are identical to those of nNOS isolated from stably transfected human kidney 293 cells. It binds N'0-nitroarginine dependent on the presence of bound tetrahydrobiopterin and exhibits a Kd of 45 nM. The enzyme is completely functional; the specific activity is 450 nmol/min per mg. This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of active nNOS for use in mechanistic and structure/function studies, as well as for drug design and development.

Chimeric Enzymes of Cytochrome P450 Oxidoreductase and Neuronal Nitric-oxide Synthase Reductase Domain Reveal Structural and Functional Differences

“…Spectral measurement of the binding constant for arginine (K s ) yielded values comparable with that of wildtype NOS (717 nM; Ref. 24). Shown in Fig.…”

“…CO difference spectra were performed as described (24). The molar protein concentrations for heme-containing chimeras were determined based on heme content via reduced CO difference spectra, where ⑀ ϭ 100 mM Ϫ1 cm Ϫ1 for ⌬A .…”

“…nNOS, CYPOR, and chimeric proteins were expressed and purified as previously described (24) except that cultures were grown in 500 ml of medium in Fernbach flasks, all of the column equilibration and wash buffers contained 100 mM NaCl, and the protein was eluted with buffer containing 500 mM NaCl and 15 mM 2Ј,3Ј-AMP. The reductase proteins (CYPOR, C FMN /nNOS FAD,NADPH , and C FMN /nNOS FAD,NADPH (tr1)) were fully oxidized by ferricyanide titration before gel filtration chromatography.…”

“…nNOS ox,FMN /C FAD,NADPH -The codons encoding the initial 963 amino acids of nNOS, encompassing the heme domain and FMN binding subdomain, were amplified by PCR using nNOS pCW (24) as template, with the following primers: Upstream, tag cta cat atg gct gaa gag aac acg; Downstream, ctt cca gga tcg atc att gct aat gag gga gtt. The forward primer encodes a NdeI site for insertion into the vector.…”

Molecular architecture of mammalian nitric oxide synthases

“…Rat nNOS in the pCWori vector was a gift of Richard B. Silverman (Northwestern University, Evanston, IL). nNOS was expressed and purified as previously described (55,56). Protein purity was assessed by SDS/PAGE and concentrations were determined by using the method of Bradford (57) with BSA as the standard.…”