Overexpression and purification of the vanilloid receptor in yeast (Saccharomyces cerevisiae).
The vanilloid receptor type 1 (VR1) is a novel membrane receptor activated by heat or chemical ligands conveying information about chemosensitive and thermosensitive pain. We have overexpressed and purified wild type VR1 (wtVR1) as well as several mutant forms using the yeast strain Saccharomyces cerevisiae with the goal of obtaining sufficient protein for structural studies. To facilitate the rapid assaying of protein production and purification we used PCR to construct mutant VR1-green fluorescent protein fusion genes. All recombinant inserts were engineered with 12 HIS tags on the C-terminus for metal affinity column purification. The yield of purified protein from 16L fermentation was about 1mg following a single-step purification procedure. By taking advantage of the calcium permeability of VR1 we measured changes in [Ca(2+)](i) in capsaicin-stimulated fura-2 loaded yeast cells expressing VR1.
D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D011955:Receptors, Drug / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D008164:Luminescent Proteins; D011955:Receptors, Drug; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins