Recombinant endothelial nitric oxide synthase: post-translational modifications in a baculovirus expression system.
Nitric oxide synthesized by the endothelial isoform of nitric oxide synthase (ecNOS) is importantly involved in the homeostatic control of blood pressure and platelet aggregation. The different members of the nitric oxide synthase protein family have several biochemical features in common but serve distinct physiological functions and are the products of distinct genes. The ecNOS is further distinguished by its subcellular distribution in the endothelial cell membrane, and the enzyme undergoes several post-translational modifications, including myristoylation, palmitoylation, and phosphorylation. Overall, however, the ecNOS has remained less well characterized because of the challenges involved in isolating sufficient quantities of this membrane-associated protein from native or cultured endothelial cells. In this report, we describe the purification and characterization of ecNOS expressed in a heterologous system in recombinant baculovirus-infected insect Sf9 cells. Recombinant ecNOS is targeted to the Sf9 cell membrane and comprises approximately 10% of the total cellular protein, allowing purification to homogeneity in a single-step procedure to yield a stable protein that retains the essential features of the native enzyme. Using biosynthetic labeling and immunoprecipitation, we show that recombinant ecNOS is myristoylated, palmitoylated, and phosphorylated when expressed in insect Sf9 cells. The interpretation of structural and enzymological studies of recombinant ecNOS will be facilitated by the apparent fidelity of its biosynthesis and post-translational modification in insect Sf9 cells.
Busconi
L
L
Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School Boston, Massachusetts 02115, USA.
Michel
T
T
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis* / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D019001:Nitric Oxide Synthase; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera
D011994:Recombinant Proteins; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases