A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel.
The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.
Department of Physiology, University of Alberta, 7-29 Medical Sciences Building, Edmonton, AB, Canada T6G 2H7.
Qiang
Q
Li
Xiao-Qing
XQ
Dai
Patrick Y
PY
Shen
Horacio F
HF
Cantiello
Edward
E
Karpinski
Xing-Zhen
XZ
Chen
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004285:Dogs; D019084:Fluorescent Antibody Technique, Indirect; D007473:Ion Channels / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000502:physiology*; D051379:Mice; D041681:NIH 3T3 Cells; D018408:Patch-Clamp Techniques; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D050396:TRPP Cation Channels
D007473:Ion Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D050396:TRPP Cation Channels