A novel, generic and effective method for the rapid purification of G protein-coupled receptors.
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.
LC1-UMR 7175, Institut Gilbert Laustriat, Pôle API, Bd Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France. magnin@esbs.u-strasbg.fr
Thierry
T
Magnin
Cédric
C
Fiez-Vandal
Noëlle
N
Potier
Aline
A
Coquard
Isabelle
I
Leray
Tania
T
Steffan
Christel
C
Logez
Fatima
F
Alkhalfioui
Franc
F
Pattus
Renaud
R
Wagner
D019921:Biotinylation; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D008863:Microspheres; D008954:Models, Biological; D010843:Pichia / Q000235:genetics; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012685:Sepharose / Q000378:metabolism; D012995:Solubility; D019809:Streptavidin / Q000302:isolation & purification / Q000378:metabolism
D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D012685:Sepharose; D019809:Streptavidin; D010450:Endopeptidases; C099488:TEV protease