Cloning and Expression of Human Inducible Nitric Oxide Synthase

Abstract

Much of the excitement over nitric oxide (NO) is owing to its diverse physiologic and pathophysiologic functions. Induced NO production has been shown to have both beneficial and detrimental consequences. The inducible or high-output NO synthase (NOS) pathway was first characterized and cloned in murine macrophages (1–3). Activation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) was uniformly used in these studies to increase inducible (i) NOS expression and improve cloning efficiency. Lyons et al. (1) injected mRNA from stimulated RAW 264.7 cells into Xenopus oocytes. RNA fractions from oocytes that expressed measurable nitrite by the Griess reaction were extracted and used to create a cDNA library that was ligated into a phage vector, lambda ZAP II. DNA from phage pools was used as a template in a polymerase chain reaction (PCR) using primers from suspected cofactor-binding sites. The radiolabeled PCR product was then used to screen the cDNA library by plaque hybridization. Recognized phages were isolated and plasmids containing the inserts were rescued by helper-phage superinfection. Two overlapping clones were combined into the expression vector pGEM. In vitro transcribed cRNA from the expression vector was then inserted back into oocytes to confirm functional expression. Nitrite production did not require the presence of Ca²⁺ and was not inhibited by trifluoperazine, a calmodulin (CaM), and brain-NOS inhibitor.