Immunoaffinity Purification and Reconstitution of Human α2-Adrenergic Receptor Subtype C2 into Phospholipid Vesicles

Liitti, Sari; Matikainen, M; Scheinin, Mika; Glumoff, Tuomo; Goldman, Adrian

doi:10.1006/prep.2001.1410

Cited by 10 publications

(7 citation statements)

References 39 publications

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“…Several studies show that the stabilization of the receptor using the tight binding of a specific ligand can increase the stability of the protein during solubilization and therefore favor its functional solubilization. For example, the functional solubilization of the human a 2C adrenergic receptor was only possible in the presence of an antagonist [39]. The stability of the rat M 3 acetylcholine (muscarinic) receptor solubilized in the presence of digitonin is also dependant on pre-stabilization of the receptor by a specific ligand [40].…”

Section: Quantification Of the Yield Of Solubilizationmentioning

confidence: 98%

“…This FLAG tag is also cleavable with enterokinase [79]. As examples, the human b 2 adrenergic, CCR5, CXCR4 and a 2C adrenergic receptors [33,36,39,51,66,80] have been purified by immunoaffinity chromatography. Except for the b 2 adrenergic receptor for which two steps were necessary [66], the three other GPCRs were purified to apparent homogeneity in a single step.…”

Section: Immunoaffinity-based Chromatographymentioning

confidence: 99%

“…The specific antibody can be directed either against the receptor [39,[68][69][70][71][72][73][74] or against a sequence fused to the recombinant receptor. Several tags are often used as epitopes for immunoaffinity purification, such as the rhodopsin tag, which consists of the C-terminal 9 amino acids of bovine rhodopsin (also known as the rho tag), [33,36,75,76], or the FLAG tag, which is the 8-9 amino acid leader peptide of the gene-10 product from bacteriophage T7 [26,43,48,61,62,77,78].…”

Section: Immunoaffinity-based Chromatographymentioning

confidence: 99%

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Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

Sarramegna

Muller

Milon

et al. 2006

Cell. Mol. Life Sci.

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G protein-coupled receptors (GPCRS) represent a class of integral membrane proteins involved in many biological processes and pathologies. Fifty percent of all modern drugs and almost 25% of the top 200 bestselling drugs are estimated to target GPCRs. Despite these crucial biological implications, very little is known, at atomic resolution, about the detailed molecular mechanisms by which these membrane proteins are able to recognize their extra-cellular stimuli and transmit the associated messages. Obviously, our understanding of GPCR functioning would be greatly facilitated by the availability of high-resolution three-dimensional (3D) structural data. However, expression, solubilization and purification of these membrane proteins are not easy to achieve, and at present, only one 3D structure has been determined, that of bovine rhodopsin. This review presents and compares the different successful strategies which have been applied to solubilize and purify recombinant GPCRs in the perspective of structural biology experiments.

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Section: Quantification Of the Yield Of Solubilizationmentioning

confidence: 98%

Section: Immunoaffinity-based Chromatographymentioning

confidence: 99%

Section: Immunoaffinity-based Chromatographymentioning

confidence: 99%

See 1 more Smart Citation

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

Sarramegna

Muller

Milon

et al. 2006

Cell. Mol. Life Sci.

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“…The expression of membrane proteins in a foreign host cell is usually not as efficient as that of soluble proteins as both the translocation to the endoplasmic reticulum membrane and the transport to their final destination are more complex than the synthesis of soluble proteins in the cytosol ( [35], for a review see [36]). Nevertheless, successful examples indicate that the yeast heterologous expression system can be useful in assessing precise functions of foreign, poorly characterized proteins [37][38][39][40][41]. Heterologous expression of chloride channels is a popular method of their investigation.…”

Section: Discussionmentioning

confidence: 99%

The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p

Flis

Hinzpeter

Edelman

et al. 2005

Biochemical Journal

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The mammalian chloride channel ClC-2 is a member of the CLC voltage-gated chloride channels family. This broadly expressed protein shows diverse cellular locations and despite numerous studies, its precise function is poorly understood. Disruption of ClC-2-encoding gene in mouse leads to retinal and testicular degeneration and mutations in CLC2 (gene encoding the ClC-2 channel) are associated with idiopathic generalized epilepsies. ClC-2 may also be responsible for Cl- transport in mouse salivary glands. The only CLC homologue of the yeast Saccharomyces cerevisiae, Gef1p, exhibits CLC activity. We expressed the mammalian ClC-2 protein in S. cerevisiae devoid of Gef1p in an attempt to identify yeast proteins influencing the functioning of ClC-2. The presence of such proteins in yeast could indicate the existence of their homologues in mammalian cells and would greatly aid their identification. Expression of ClC-2 in yeast required optimization of the sequence context of the AUG translation initiation codon. After obtaining an efficient translation, we found that rat ClC-2 cannot directly substitute for yeast Gef1p. Functional substitution for Gef1p was, however, achieved in the presence of an increased level of intact or C-terminally truncated yeast Kha1 protein. Based on the deduced amino acid sequence, the Kha1 protein can be classified as a Na+/H+ transporter since it has a large N-terminal domain similar to the family of NHEs (Na+/H+ exchangers). This suggests that the Kha1p may take part in the regulation of intracellular cation homoeostasis and pH control. We have established that Kha1p is localized in the same cellular compartment as Gef1p and yeast-expressed ClC-2: the Golgi apparatus. We propose that Kha1p may aid ClC-2-dependent suppression of the Deltagef1-associated growth defects by keeping the Golgi apparatus pH in a range suitable for ClC-2 activity. The approach employed in the present study may be of general applicability to the characterization of poorly understood proteins by their functional expression in yeast.

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“…The abundant proteins belonging to the superfamily of G-protein coupled receptors (GPCR) that possess seven transmembrane a helices, have important roles in eukaryotic signalling. Few GPCR have been reconstituted, by a variety of coinsertion or postinsertion protocols, with variable results [4,[15][16][17]. The b-chemokines RANTES, MIP-1a, and MIP-1b are recognized by the GPCR CCR5 [18][19][20][21].…”

mentioning

confidence: 99%

Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes

Devesa

Chams

Dinadayala

et al. 2002

European Journal of Biochemistry

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Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical properties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane a helices and small ecto-and endodomains. A His 6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-b-D-maltoside (DDM) and reconstituted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polystyrene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins.

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Immunoaffinity Purification and Reconstitution of Human α2-Adrenergic Receptor Subtype C2 into Phospholipid Vesicles

Cited by 10 publications

References 39 publications

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

Recombinant G protein-coupled receptors from expression to renaturation: a challenge towards structure

The functioning of mammalian ClC-2 chloride channel in Saccharomyces cerevisiae cells requires an increased level of Kha1p

Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes

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