Production and purification of large quantities of the functional N-terminal ectodomain of human VPAC1 receptor.
Vasoactive intestinal peptide (VIP) is implicated in many physiological and pathophysiological processes, and its receptors are promising targets for the development of new drugs. The human VPAC1 receptor for VIP and pituitary adenylate cyclase-activating polypeptide is a class II G protein coupled receptor. The N-terminal ectodomain (N-ted) of the VPAC1 receptor is a major VIP binding site. To determinate the high resolution structure of the VPAC1 receptor N-ted, large quantities of purified recombinant N-ted produced are required. The N-ted sequence (31-144), which is fused to thioredoxin protein and 6xHis tag, was expressed into Origami Escherichia coli strain. Purification of recombinant N-ted using Ni-NTA affinity column associated to Nu-polyacrylamide gel electrophoresis analysis reveals the presence of one single band of Mw 19,000 corresponding to the purified recombinant N-ted. The purified N-ted was able to recognize VIP and the selective antagonist PG96-269. About 5-10 mg of functional purified protein/liter of bacterial culture is currently produced. This is a crucial step to determine the structure of functional human VPAC1 receptor N-ted by nuclear magnetic resonance.
Couvineau
Alain
A
INSERM U773, Institut National de la Santé et de la Recherche Médicale, Centre de Recherche Biomédicale Bichat-Beaujon, CRB3, Faculté de Médecine Xavier Bichat, 75018, Paris, France. alain.couvineau@inserm.fr
Robert
Jean-Claude
JC
Ramdani
Tarik
T
Lacapère
Jean-Jacques
JJ
Rouyer-Fessard
Christiane
C
Laburthe
Marc
M
D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D011487:Protein Conformation; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D014660:Vasoactive Intestinal Peptide / Q000235:genetics / Q000378:metabolism*
D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D014660:Vasoactive Intestinal Peptide