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Expression and Purification of G-protein coupled receptors
Beta2-adrenergic receptor Ghanouni et al., 2000:
- epitope tagged with influenza-hemagglutinin signal sequence followed by the FLAG epitope and tagged at the C-terminus with 6 His
- baculovirus pVL1392
- Sf9 cells with BaculoGold tranfection
- Yield: 5 nmol of pure protein from 1 liter (in Jensen et al, JBC2001, 1-2 nmol per 500 ml culture)
- 1% NDM, n-dodecyl-beta-D-maltoside,
- not very stable, ligand binding goes down from 1 hour to 3 hours significantly
- also check reference Gether, U., J.A. Lowe, 3rd, and T.W. Schwartz, Tachykinin non-peptide antagonists: binding domain and molecular mode of action. Biochem Soc Trans, 1995. 23(1): p. 96-102.
- Rasmussen, S. G., A. D. Jensen, et al. (1999). "Mutation of a highly conserved aspartic acid in the beta2 adrenergic receptor: constitutive activation, structural instability, and conformational rearrangement of transmembrane segment 6." Mol Pharmacol 56(1): 175-84. partially purified receptor protien indetergen tsolution at 37oC, wehere T1/2 is 12-24 hours
- Peleg, G., P. Ghanouni, B.K. Kobilka, and R.N. Zare, Single-molecule spectroscopy of the beta(2) adrenergic receptor: observation of conformational substates in a membrane protein. Proc Natl Acad Sci U S A, 2001. 98(15): p. 8469-74: in N-dodecyl-b-D-maltoside
Delta-opioid receptor Salamon et al., 2000
- myc epitope at C-term
- pcDNA3 vector with myc/His tag
- expression in Cho cells
- purification on Talon Co2+ metal chelating column
- elution in 30mM octyl glucoside
PACAP (pituitary adenylate cyclase activating polypeptide) receptor (Class B receptor)
- in Sf9 cells
- solubilized with digitonin
- purified using affinity chromatography according to ref. Ohtaki, T. and e. al., J Biol Chem, 1998. 273: p. 15464-15473
Abdulaev, N.G. and K.D. Ridge, Heterologous expression of bovine opsin in Pichia pastoris. Methods Enzymol, 2000. 315: p. 3-11.
Expression and Purification of G-proteins
- from Sf9 cells with or without His-tag (Sheikh et al., 1999 and ref. therein)