Functional expression and purification of histidine-tagged rat renal Na/Phosphate (NaPi-2) and Na/Sulfate (NaSi-1) cotransporters.
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/Pi-cotransporter exhibited more than 60-fold higher sodium-dependent transport of phosphate compared to noninfected cells. Expressed Na/Pi-cotransport exhibited a Km of Pi of 0.21 mm and an apparent Km 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 Km of SO2-4 of about 0.3-0.4 mm and a Km of sodium of 55 mm. By Western blotting and immunoprecipitation expressed NaSi-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.
Fucentese
M
M
Institute of Physiology, University of Zürich, Winterthurerstr. 190, CH-8057, Zürich, Switzerland.
Winterhalter
K H
KH
Murer
H
H
Biber
J
J
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D027682:Cation Transport Proteins; D002460:Cell Line; D015870:Gene Expression; D017136:Ion Transport; D007668:Kidney / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000077003:Sodium Sulfate Cotransporter; D050608:Sodium-Phosphate Cotransporter Proteins; D012995:Solubility; D018411:Spodoptera; D013431:Sulfates / Q000378:metabolism; D027981:Symporters
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D010710:Phosphates; D011994:Recombinant Proteins; D000077003:Sodium Sulfate Cotransporter; D050608:Sodium-Phosphate Cotransporter Proteins; D013431:Sulfates; D027981:Symporters; D012964:Sodium