Stable transfection of MDCK cells with epitope-tagged human PepT1

Abstract

Purpose: Establish and characterize a MDCK cell line that stably expressed PepT1.

Methods: MDCK cells stably transfected with N-terminal HA-tagged hPepT1 were examined by immunostaining using antibodies to HA and hPepT1. HA-hPepT1 expression was verified by immunoprecipitation and Western blotting from cell lysates. The function of the expressed HA-hPepT1 was evaluated by glycyl-sarcosine uptake. The half-life of PepT1 on the cell surface was measured by biotinylation and chase-labeling of the cells, followed by immunoprecipitation with the anti-PepT1 antibody and blotting with HRP-streptavidin.

Results: Immunolabeling with antibodies to HA and PepT1 showed fluorescence colocalization. Immunoprecipitation with the anti-HA antibody and Western blotting with the anti-PepT1 antibody showed a broad 90-105 kDa band, and vice versa when the antibodies were utilized in the reverse order. Glycyl-sarcosine uptake was increased in the stably transfected cells by 2-5 fold over untransfected control cells. The Km value of 375 microM accurately reflected the characteristic low affinity of PepT1. Finally, the half-life of the surface biotinylated HA-hPepT1 was measured to be 22 hrs.

Conclusions: A MDCK cell line stably expressed intact and functional HA-tagged hPepT1 on its cell surface. This cell line represents an alternative to the use of Caco-2 cells to evaluate PepT1 function.