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.
Magnin
Thierry
T
LC1-UMR 7175, Institut Gilbert Laustriat, Pôle API, Bd Sébastien Brant, BP 10413, 67412 Illkirch Cedex, France. magnin@esbs.u-strasbg.fr
Fiez-Vandal
Cédric
C
Potier
Noëlle
N
Coquard
Aline
A
Leray
Isabelle
I
Steffan
Tania
T
Logez
Christel
C
Alkhalfioui
Fatima
F
Pattus
Franc
F
Wagner
Renaud
R
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