Overproduction of neuronal nitric oxide synthase

Details Overproduction of neuronal nitric oxide synthase Overproduction of neuronal nitric oxide synthase

1995-08-24

1999-07-06

Wax, Robert A.

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Oxidoreductase

435 691, 4352523, 43525233, 536 232, C12N 902, C12N 1553, C12N 1570

Patent

active

059196823

ABSTRACT:
The present invention is directed to overproduction of nitric oxide synthase (NOS) in a prokaryote. More particularly, the invention involves overexpression of functional neuronal NOS in a protease-deficient strain of Escherichia coli, utilizing a pCW vector under the control of the tac promotor. The invention further involves co-expression of the protein with folding agonists, or chaperonins. The enzyme produced is complete with heme and flavins, and may be activated by incubation with tetrahydrobiopterin. It may be isolated as a predominantly high spin heme protein that demonstrates spectral properties which are identical to those of nNOS isolated from human kidney 293 cells. The methods disclosed are contemplated to be useful in expressing large amounts of other nitric oxide synthases, as well as other proteins that are difficult to produce correctly folded in prokaryotes.

REFERENCES:
patent: 5240831 (1993-08-01), Barnes
patent: 5268465 (1993-12-01), Bredt et al.
Makrides, 60(3) Microbiol. Rev. 512-538 (1996).
Lee et al., 267(5) J. Biol. Chem. 2849-52 (1992).
Studier et al., 185 Meth. Enz. 60-89 (1990).
Barnes et al., "Expression and enzymatic activity of recombinant P450 17.alpha.-hydroxylas in Escherichia coli", Proc. Natl. Acad. Sci. USA, 88:5597-5601, Jul. 1991.
Bredt et al., "Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase", Nature, 351:714-718, Jun. 1991.
Bredt, David S. and Solomon H. Snyder, "Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme", Proc. Natl. Acad. Sci. USA, 87:682-685, Jan. 1990.
Bredt, D.S. and S.H. Snyder, "Nitric Oxide: A Physiologic Messenger Molecule", Ann. Rev. Biochem., 63:175-195, 1994.
Busconi, Liliana and Thomas Michel, "Recombinant Endothelial Nitric Oxide Synthase: Post-translational Modifications in a Baculovirus Expression System", Molecular Pharmacology, 47:655-659, 1995.
Charles et al., "Cloning and Expression of a Rat Neuronal Nitric Oxide Synthase Coding Sequence in a Baculovirus/Insect Cell System", Biochemical and Biophysical Research Communications, 196(3):1481-1489, Nov. 1993.
Cho et al., "Calmodulin Is a Subunit of Nitric Oxide Synthase from Macrophages", J. Exp. Med, 176:599-604, Aug. 1992.
Forstermann et al., "Calmodulin-dependent endothelium-derived relaxing factor
itric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells", Proc. Natl. Acad. Sci. USA, 88:1788-1792, Mar. 1991.
Fukushima, Takeshi and Jon C. Nixon, "Analysis of Reduced Forms of Biopterin in Biological Tissues and fluids", Analytical Biochemistry, 102:176-188, 1980.
Fukuto, J.M. and G. Chaudhuri, "Inhibition of Constitutive and Inducible Nitric Oxide Synthase: Potential Selective Inhibition", Annu. Rev. Pharmacoi Toxical, 35:165-194, 1995.
Gegner, Julie A. and Frederick W. Dahlquist, "Signal transduction in bacteria: CheW forms a reversible complex with the protein kinase CheA", Proc. Natl. Acad. Sci. USA, 88:750-754, Feb. 1991.
Geller et al., "Molecular Cloning and Expression of Inducible Nitric Oxide Synthase From Human Hepatocytes", Proc. Natl. Acad. Sci USA, 90:3491-3495, Apr. 1993.
Gerber, N.C. and P.R. Ortiz de Montellano, "Active Site Topology of Nitric Oxide Synthase Expressed in E. Coli", FASEB J. 9, A1490 (Abstract #1552).
Gerber, N.C. and P.R. Ortiz de Montellano, "Neuronal Nitrate Oxide Synthase, Expression in Escherichia coli, Irreversible Inhibition by Phenylidiazene, and Active Site Topology", The Journal of Biological Chemistry, 270(30):17791-17796, Jul. 1995.
Goloubinoff et al., GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli, 337:44-47.
Gross, Steven S. and Roberto Levi, "An Absolute Requirement for Cytokine-Induced Nitric Oxide Generation by Vascular Smooth Muscle", The Journal of Biological Chemistry, 267(36)25722-25729.
Hevel et al., Purification of the Inducible Murine Macrophage Nitric Oxide Synthase, The Journal of Biological Chemistry, 266(34):22789-22791.
Janssens et al., "Cloning and Expression of a cDNA Encoding Human Endothelium-Derived Relaxing Factor/Nitric Oxide Synthase", The Journal of Biological Chemistry, 267(21):14519-14522, 1992.
Kelm, Malte and Jugen Schrader, "Control of Coronary Vascular Tone by Nitric Oxide", Circulation Research, 66(6):1561-1575, Jun. 1990.
Kim et al., "Cystosolic chaperonin subunits have a conserved ATPase domain but diverged polypeptide-binding domains", TIBS 19, 1543-548, Dec. 1994.
Klatt et al., "Brain nitric oxide synthase is a haemoprotein", Biochem J., 288:15-17, 1992.
Kobzik et al., "Nitric Oxide in Skeletal Muscle", Nature, 372:546-548, Dec. 1994.
Lamas et al., "Endothelial nitric oxide synthase: Molecular cloning and characterization of a distinct constitutive enzyme isoform", Proc. Natl. Acad. Sci. USA, 89:6348-6352, Jul. 1992.
Lowenstein et al., "Cloned and expressed macrophage nitric oxide synthase contrasts with the brain enzyme", Proc. Natl. Acad. Sci. USA, 89:6711-6715.
Michael A. Marletta, "Nitric Oxide Synthase Structure and Mechanism", The Journal of Biological Chemistry, 268(17):12231-12234, 1993.
Martinus et al., "Role of chaperones in the biogenesis and maintenance of the mitochondrion", The FASEB Journal, 9:371-378.
Bettie Sue Siler Masters, "Nitric Oxide Synthases: Why So Complex"? Annu. Rev. Nutr., 14:131-145, 1994.
Mayer et al., "Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase", FEBS Letters, 288(1,2):187-191, Aug. 1991.
McMillan et al., "Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide", Proc. Natl. Acad. Sci. USA, 89:11141-11145, Dec. 1992.
McMillian et al., "Prokaryotic Expression of the Heme- and Flavin-Binding Domains of Rat Neuronal Nitric Oxide Synthase as Distinct Polypeptides: Identification of the Heme-Binding Proximal Thiolate Ligand as Cysteine-415", Biochemistry, 34:3686-3693, 1995.
McMillan, Kirk and Bettie Sue Siler Masters, "Optical Difference Spectrophotometry as a Probe of Rat Brain Nitric Oxide Synthase Heme-Substrate Interaction", The American Chemical Society, 32(38):9875-9880, Sep., 1993.
Nakane et al., "Cloned Human Brain Oxide Synthase is Highly Expressed in Skeletal Muscle", Federation of European Biochemical Societies, 316(2):175-180, Jan. 1993.
Nelson et al., "The P450 Superfamily: Update on New Sequences, Gene Mapping, Acession Numbers, Early Trivial Names of Enzymes, and Nomenclature", DNA and Cell Biology, 12(1):1-51, 1993.
Ogden J.E. and P.K. Moore, "Inhibition of Nitric Oxide Synthase--Potential for a Novel Class of Therapeutic Agent"? TBTECH, 13:70-78, Feb. 1995.
Pollock et al., Purification and characerization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells, Proc. Natl. Acad. Sci. USA, 88:10480-10484, Dec. 1991.
Richards M.K. and M.A. Marletta, "Characterization of Neuronal Nitric Oxide Synthase and a C415H Mutant, Purified from a Baculovirus Overexpression System", Biochemistry, 33:14723-14732, 1994.
J. S. Rieske, The Quantitative Determination of Mitochondrial Hemoproteins, Methods Enzymol., 10:488-493, 1967.
Riveros-Moreno et al., "Purification to homogeneity and characerization of rat brain recombinant nitric oxide synthase", Eur. J. Biochem., 230:52-57, 1995.
Ca.sup.2+ /Calmodulin-Dependent NO Synthase Type I: A Bioteroflavoprotein with Ca.sup.2+ /Calmodulin-independent Diaphorase and Reductase Activities, Biochemistry, 31(12):3243-3249, 1992.
Schmidt et al., "Purification of a soluble isoform of guanylyl cyclase-activating-factor synthase", Proc. Natl. Acad. Sci. USA, 88:365-369, Jan. 1991.
Seo et al., "Heme Requirement for Production of Active Endothelial Nitric Oxide Synthase in Baculovirus-Infected Insect Cells", Biochemical and Biophysical Research Communications, 208(1):10-18, Mar. 1995.
Sessa et al., "Molecular cloning and Expression of a cDNA Encoding Endothelial Cell Nitric Oxide Synthase", The Journal of Biological Chemistry, 22:15274-15

Affiliated with

Also associated with

Rating

Overproduction of neuronal nitric oxide synthase does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Overproduction of neuronal nitric oxide synthase, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Overproduction of neuronal nitric oxide synthase will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-898600