ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005


The Latest on X

Kidney Week

Please note that you are viewing an archived section from 2022 and some content may be unavailable. To unlock all content for 2022, please visit the archives.

Abstract: TH-PO315

Stable Transduction of Recombinant NHERF1 Protein in Opossum Kidney Cells

Session Information

Category: Fluid‚ Electrolyte‚ and Acid-Base Disorders

  • 1001 Fluid‚ Electrolyte‚ and Acid-Base Disorders: Basic


  • Gagnon, Kenneth Bradley, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Rane, Madhavi J., University of Louisville School of Medicine, Louisville, Kentucky, United States
  • Lederer, Eleanor D., The University of Texas Southwestern Medical Center, Dallas, Texas, United States

NHERF1 (Na+-hydrogen exchanger regulatory factor isoform 1) is a cytoplasmic scaffolding protein containing two internal postsynaptic density 95/disc large/zona occludens binding domains and a carboxy-terminal ezrin-binding domain. NHERF1 anchors the Na+-dependent phosphate cotransporter type IIa (Npt2a) in the brush border membrane of proximal tubules through its cross-linking of Npt2a with the actin-filament binding protein, Ezrin. The opossum kidney (OK) cell line, originally derived from a female Virginia Opossum (Virginia delphi), has been extensively used as a model of proximal tubular epithelia. A sub-clone of the OK cell line (OK-H) expresses low levels of endogenous NHERF1 and lacks PTH-mediated inhibition of sodium-dependent phosphate uptake.


We PCR cloned full-length mouse NHERF1 into a bacterial expression vector and transformed BL21 E. coli to over-express the HIS-tagged recombinant NHERF1 (rmNHERF1) protein. We purified rmNHERF1 utilizing the upstream poly-histidine tag and immobilized metal affinity chromatography. We added purified rmNHERF1 to OK-H cells at different concentrations and time points to assess the in vitro stability of the recombinant protein by SDS-PAGE and immunoblotting. We also transduced OK-H cells with rmNHERF1 for 4 and 24 h and measured radiolabeled phosphate uptake compared to non-transduced OK-H cells.


Immunoblotting whole cell lysates transduced with rmNHERF1 for 4 h with both an anti-HIS and an anti-NHERF1 antibody demonstrate a significant dose-dependent increase in rmNHERF1 expression in OK-H cells. Similarly, OK-H cells transduced with rmNHERF1 for 4, 8, 24, and 48 h demonstrates stable temporal expression of the recombinant protein with minimal lysosomal degradation. We also detected Npt2a expression in transduced OK-H cells compared to non-transduced controls. Radiolabeled phosphate uptake of OK-H cells transduced with rmNHERF1 for 4 h and 24 h did not produce a significant increase in phosphate uptake.


We have demonstrated the stable introduction of recombinant mouse NHERF1 protein in NHERF1-deficient opossum kidney cells with a resulting increase in Npt2a expression but not concurrent increase in phosphate uptake activity suggesting that the recombinant fusion protein may be inert or trapped within an intracellular vesicle following endocytic transduction.


  • Veterans Affairs Support