Expression of Functional Na+/K+-ATPase in Insect Cells Using Baculovirus

Abstract

Expression systems for the Na+/K+-ATPase have been hindered because most commonly used cell lines have high endogenous levels of the Na pump. In contrast, some insect cells have little or no Na+/K+-ATPase activity. Recently, the insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV) has been adapted for the overproduction of recombinant proteins in insect cells. In this system, a foreign gene replaces the nonessential AcNPV polyhedrin gene. The foreign gene is then under control of the polyhedrin promoter, a promoter that is extremely active late in the infective cycle. Using the baculovirus expression system a wide variety of exogenous genes have been produced in insect cells, most commonly a cell line (Sf-9) from the ovary of the fall armyworm Spodoptera frugiperda. A major advantage of this system is that the insect cells perform most of the post-translational modifications found in mammalian cells. Infected insect cells will accurately complete the glycosylation, signal peptide cleavage, proteolytic processing, phosphorylation, palmitylation and myristylation dictated by the primary structure of the polypeptide. In addition, insect cells will successfully express and assemble multimeric proteins and rigorously sort recombinant proteins to their proper cellular domains (20). Using this system, we have produced functional Na+/K+-ATPase activity in insect cells (4,8). Ouabain-sensitive ATPase activity, isolation of a sodium-dependent potassium- and ouabain-sensitive ATP phosphorylated intermediate, isolation of a ouabain-stimulated Pi phosphorylated intermediate, and ouabain-sensitive 86Rb uptake, all demonstrate that the baculovirusinduced Na+/K+-ATPase is identical to the native enzyme. In addition, because each subunit can be produced separately, we have obtained some unexpected and surprising results concerning the expression of the individual subunits.