6] Endothelial nitric oxide synthase expression in heterologous systems

Abstract

Publisher Summary This chapter discusses the endothelial nitric oxide synthase (NOS) expression in heterologous systems. These expression systems can be exploited for the analysis of enzyme structure-function relationships, and for the study of NOS biosynthesis and metabolism. The various heterologous expression systems each require that the relevant NOS complementary DNA (cDNA) be cloned into a plasmid or viral construct possessing specific features appropriate for the given expression system. The experimental principles and methodologies underlying these expression systems reflect the modification of standard protocols for the isolation and expression of generic eukaryotic cDNAs. Cell-free protein expression protocols permit the analysis of NOS in an easily manipulated in vitro system. Mutations that result in changes in protein structure or function can be generated directly by alterations in the template cDNA used for in vitro messenger RNA (mRNA) and protein synthesis. For coupled in vitro transcription and translation of endothelial cell NOS (ecNOS), the cDNA insert is cloned into the multiple cloning site of a plasmid expression vector in a “sense” orientation, downstream of the relevant promoter sequences located on the plasmid. COS-7 cells are useful in analyses of ecNOS protein expression in transient cDNA transfection experiments.