High-level expression of human inducible nitric oxide synthase in Chinese hamster ovary cells and characterization of the purified enzyme.
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.
Laubach
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Department of Cell Biology, Glaxo Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
Garvey
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Sherman
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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
D002087:Butyrates; D011994:Recombinant Proteins; D020148:Butyric Acid; D019001:Nitric Oxide Synthase