ISSN:
1432-1424
Keywords:
Key words: Renal Na/Pi-cotransport — Renal Na/sulfate-cotransport — Sf9 cells — Purification — Ni2+-chelate chromatography
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/s002329900300
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