Functional Expression and Purification of Histidine-Tagged Rat Renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) Cotransporters |
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Authors: | M Fucentese KH Winterhalter H Murer J Biber |
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Institution: | (1) Institute of Physiology, University of Zürich, Winterthurerstr. 190, CH-8057, Zürich, Switzerland, CH;(2) Laboratory of Biochemistry I, ETH-Zürich, 8057 Zürich, Switzerland, CH |
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Abstract: | Two mammalian sodium-dependent anion-cotransporters (NaPi-2 for phosphate and NaSi-1 for sulfate) have been expressed in
Sf9 insect cells using the baculovirus expression system. A histidine tag was introduced at the C-termini in order to facilitate
purification by metal-affinity chromatography. Sf9 cells infected with the histidine-tagged Ni/P
i
-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed
Na/P
i
-cotransport exhibited a K
m
of P
i
of 0.21 mm and an apparent K
m
of sodium of 92 mm. Infected cells expressed a 65 kDa polypeptide as detected by Western blotting and immunoprecipitation. Sf9 cells infected
with the histidine-tagged NaSi-1 or untagged NaSi-1 protein expressed sodium-dependent sulfate cotransport up to 60-fold higher
compared to noninfected cells. Transport of sulfate was highly dependent on sodium exhibiting a K
m
of SO2−
4 of about 0.3–0.4 mm and a K
m
of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaS
i
-1 proteins were detected at 55–60 kDa. These studies demonstrate that histidine tagged proximal tubular Na-dependent cotransporters
for phosphate and sulfate can be expressed functionally in Sf9 cells and that the kinetic characteristics were not altered
by the introduction of a histidine tag at the C-termini. Furthermore, it is demonstrated that after solubilization under denaturing
conditions histidine-tagged cotransporter proteins can be purified by metal-chelate affinity chromatography.
Received: 24 March 1997/Revised: 8 July 1997 |
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Keywords: | : Renal Na/Pi-cotransport — Renal Na/sulfate-cotransport — Sf9 cells — Purification — Ni2+-chelate chromatography |
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