Expression of the Na/P(i)-cotransporter type IIb in Sf9 cells: functional characterization and purification.

Abstract

In mammals the type IIb Na/P(i)-cotransporter is expressed in various tissues such as intestine, brain, lung and testis. The type IIb cotransporter shows 51% homology with the renal type IIa Na/P(i)-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/P(i)-cotransporter available for future structural studies, we have expressed this cotransporter in Sf9 cells. Sf9 cells were infected with recombinant baculovirus containing 6His NaPi-IIb. Infected cells expressed a polypeptide of approximately 90 kDa, corresponding to a partially glycosylated form of the type IIb cotransporter. Transport studies demonstrated that the type IIb protein expressed in Sf9 cells mediates transport of phosphate in a Na-dependent manner with similar kinetic characteristics (apparent K(m)s for sodium and phosphate and pH dependence) as previously described. Solubilization experiments demonstrated that, in contrast to the type IIa cotransporter, the type IIb can be solubilized by nonionic detergents and that solubilized type IIb Na/P(i)-cotransporter can be purified by Ni-NTA chromatography.