High-level expression of functional rat neuronal nitric oxide synthase in Escherichia coli.
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 of E. 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 omega-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.
Roman
L J
LJ
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760, USA.
Sheta
E A
EA
Martasek
P
P
Gross
S S
SS
Liu
Q
Q
Masters
B S
BS
D000594:Amino Acid Oxidoreductases / Q000235:genetics*; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D018835:Chaperonin 10 / Q000235:genetics; D018834:Chaperonin 60 / Q000235:genetics; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics*; D006868:Hydrolysis; D008297:Male; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D019335:Nitroarginine; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011622:Pterins / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013057:Spectrum Analysis
D018835:Chaperonin 10; D018834:Chaperonin 60; D017931:DNA Primers; D018076:DNA, Complementary; D011622:Pterins; D011994:Recombinant Proteins; D019335:Nitroarginine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases