title: High-level expression of human inducible nitric oxide synthase in Chinese hamster ovary cells and characterization of the purified enzyme.
abstract: Chinese hamster ovary cells were transfected with the human inducible nitric oxide synthase (iNOS) cDNA under the control of the hCMV promoter. The NOS inhibitor, S-ethyl isothiourea, was utilized to minimize toxicity from NO production. Selected colonies were grown in the absence of S-ethyl isothiourea, after which nitrite levels in the medium were measured. NOS activities in cell lysates were determined, and 9 colonies had iNOS activities of over 1 nmol of citrulline formed/min/mg protein. iNOS expression was further increased by gene amplification and the use of sodium butyrate, resulting in two cell lines with stable activity of greater than 3 nmol/min/mg. The iNOS purified from these cells had a specific activity of over 500 nmol/min/mg, and its properties were similar to native iNOS purified from cytokine-induced DLD-1 cells. This is the most efficient expression system reported to date for iNOS.
authors.affiliation: Department of Cell Biology, Glaxo Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
authors.forename: V E
authors.initials: VE
authors.lastname: Laubach
authors.forename: E P
authors.initials: EP
authors.lastname: Garvey
authors.forename: P A
authors.initials: PA
authors.lastname: Sherman
mesh_terms: D000818:Animals; D002087:Butyrates / Q000494:pharmacology; D020148:Butyric Acid; D016466:CHO Cells; D006224:Cricetinae; D004790:Enzyme Induction; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins
chemical_list: D002087:Butyrates; D011994:Recombinant Proteins; D020148:Butyric Acid; D019001:Nitric Oxide Synthase