Rho Antibody And Tag To Purify Cell Surface Proteins
A process for the expression and purification of membrane associated proteins is provided. A process for the expression and purification of membrane proteins such as G-protein coupled receptors is provided. A hybrid polypeptide is constructed from a protein of interest and a peptide tag from the amino-terminus extracellular domain of rhodopsin. A method for purifying the hybrid protein using the rhodopsin tag is provided. Methods of detecting the expressed hybrid are also provided.
1 . A hybrid polypeptide comprising:
a target polypeptide,
an identification peptide comprising the amino-terminal extracellular domain of rhodopsin, where said identification peptide is linked to the amino-terminus of said target polypeptide.
2 . The hybrid polypeptide of claim 1 where said identification peptide comprises an amino terminal extracellular domain of bovine rhodopsin.
3 . The hybrid polypeptide of claim 2 where said identification peptide comprises amino-terminal amino acids of bovine rhodopsin selected from the group consisting of: a first 20, a first 25, a first 30, and a first 35 amino acids.
4 . The hybrid polypeptide of claim 1 where said target polypeptide comprises a membrane protein.
5 . The hybrid polypeptide of claim 4 where said target polypeptide comprises a G-protein coupled receptor protein.
6 . A method of purifying a target polypeptide comprising:
expressing a hybrid polypeptide comprising an identification peptide of the amino terminal extracellular domain of rhodopsin fused to the amino terminal region of a target polypeptide,
forming a complex of said hybrid polypeptide with an antibody against said amino terminal domain of rhodopsin,
isolating said complex, and
dissociating said hybrid polypeptide from said antibody.
7 . The method of claim 6 where said target polypeptide comprises a G-protein coupled receptor.
8 . The method of claim 6 where said identification peptide comprises amino-terminal amino acids of bovine rhodopsin selected from the group consisting of: a first 20, a first 25, a first 30, and a first 35 amino acids.
9 . The method of claim 8 where said antibody is directed against a first 15 amino acids of bovine rhodopsin.
10 . The method of claim 6 where said antibody is held by a resin.
11 . The method of claim 6 where said hybrid polypeptide is dissociated from said antibody by a pH gradient
12 . The method of claim 6 where said hybrid polypeptide is dissociated from said antibody by a salt gradient.
13 . The method of claim 6 where said hybrid polypeptide is dissociated from said antibody by competing with a peptide comprising of all or a portion of the amino terminal domain of rhodopsin.
14 . A method of labeling a target polypeptide comprising:
expressing a hybrid polypeptide comprising: an amino terminal extracellular domain of rhodopsin fused to an amino terminal region of a target polypeptide;
forming a complex of said hybrid polypeptide and an antibody against said amino terminal extracellular domain of rhodopsin; and
detecting said complex by means of a reporting agent.
15 . The method of claim 14 where said amino terminal domain of rhodopsin comprises amino-terminal amino acids of bovine rhodopsin selected from the group consisting of: a first 20, a first 25, a first 30, and a first 35 amino acids.
16 . The method of claim 15 where said antibody is directed against a first 15 amino acids of bovine rhodopsin.
17 . The method of claim 14 where said target polypeptide is a G-protein coupled receptor.
18 . The method of claim 14 where said reporting agent is conjugated to said antibody.
19 . The method of claim 14 where said reporting agent is a fluorescent marker.
20 . The method of claim 14 where said reporting agent is an enzymatic marker.
21 . The method of claim 14 where said reporting agent is a radioactive marker.
22 . The method of claim 14 where said complex is detected by biding said complex to a secondary antibody against said antibody to the amino terminal extracellular domain of rhodopsin, and said secondary antibody is linked to a reporting agent.