10] Heterologous expression of mammalian P450 in COS cells

Abstract

This chapter discusses the heterologous expression of mammalian P450 in COS Cells. Heterologous expression systems are employed to study structural and functional aspects of various forms of cytochrome P450 has. A primary requirement for such an expression system is that it has low background levels of endogenous P450. The purpose of this chapter is to describe potential uses and limitations of transient transfection of COS cells to study P450 function and structure. The focus is on transient transfection of COS cells, subcellular fractionation of transfected COS 1 cells, measurement of P450 activity, and quantitation of P450 expression. The optimal method for transfection of mammalian cells with plasmid DNA is dependent on the cell type. Calcium phosphate precipitation, DEAE-dextran, electroporation, and lipofection have all been successfully used with COS 1 cells. The COS 1 expression system is useful in studies to identify, confirm, or assign the specific enzymatic activities to cloned P450 complentary DNA (cDNAs).