Expression of the Na/Pi-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

Expression of the Na/Pi-cotransporter Type IIb in Sf9 Cells: Functional Characterization and... In mammals the type IIb Na/Pi-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/Pi-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/Pi-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 ~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 ms 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/Pi-cotransporter can be purified by Ni-NTA chromatography. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Expression of the Na/Pi-cotransporter Type IIb in Sf9 Cells: Functional Characterization and Purification

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Publisher
Springer-Verlag
Copyright
Copyright © 2003 by Springer-Verlag New York Inc.
Subject
Philosophy
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-003-2028-6
Publisher site
See Article on Publisher Site

Abstract

In mammals the type IIb Na/Pi-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/Pi-cotransporter, for which a detailed model of the secondary structure has emerged based on recent structure/function studies. To make the type IIb Na/Pi-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 ~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 ms 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/Pi-cotransporter can be purified by Ni-NTA chromatography.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Sep 18, 2003

References

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