Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris

Sarramegna, Valérie; Muller, Isabelle S.; Mousseau, Guillaume; Froment, Carine; Monsarrat, Bernard; Milon, Alain; Talmont, Franck

doi:10.1016/j.pep.2005.05.007

Cited by 44 publications

(46 citation statements)

References 33 publications

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“…Reconstitution of MOR required the purification of active receptor in large enough quantities for subsequent biochemical manipulation and analysis. Previous reports of MOR purification from endogenous or recombinant sources have yielded either low quantities (38 -42) or poor agonist binding affinities (43)(44)(45). Several aspects were key to the expression and purification of YFP-MOR (YMOR): a cleavable hemagglutinin signal sequence at the N terminus of the receptor (46), the presence of naltrexone during expression, and the inclusion of cholesteryl hemisuccinate and naltrexone during the entire purification process.…”

Section: Discussionmentioning

confidence: 99%

Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors

Kuszak

Pitchiaya

Anand

et al. 2009

Journal of Biological Chemistry

162

155

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Despite extensive characterization of the -opioid receptor (MOR), the biochemical properties of the isolated receptor remain unclear. In light of recent reports, we proposed that the monomeric form of MOR can activate G proteins and be subject to allosteric regulation. A -opioid receptor fused to yellow fluorescent protein (YMOR) was constructed and expressed in insect cells. YMOR binds ligands with high affinity, displays agonist-stimulated [35 S]guanosine 5-(␥-thio)triphosphate binding to G␣ i , and is allosterically regulated by coupled G i protein heterotrimer both in insect cell membranes and as purified protein reconstituted into a phospholipid bilayer in the form of high density lipoprotein particles. Single-particle imaging of fluorescently labeled receptor indicates that the reconstituted YMOR is monomeric. Moreover, single-molecule imaging of a Cy3-labeled agonist, [Lys 7 , Cys 8 ]dermorphin, illustrates a novel method for studying G protein-coupled receptor-ligand binding and suggests that one molecule of agonist binds per monomeric YMOR. Together these data support the notion that oligomerization of the -opioid receptor is not required for agonist and antagonist binding and that the monomeric receptor is the minimal functional unit in regard to G protein activation and strong allosteric regulation of agonist binding by G proteins.Opioid receptors are members of the G protein-coupled receptor (GPCR) 2 superfamily and are clinical mainstays for inducing analgesia. Three isoforms of opioid receptors, , ␦, and , have been cloned and are known to couple to G i/o proteins to regulate adenylyl cyclase and K ϩ /Ca ϩ ion channels(1-3). An ever growing amount of data suggests that many GPCRs oligomerize (4, 5), and several studies have suggested that -opioid receptors (MORs) and ␦-opioid receptors heterodimerize to form unique ligand binding and G protein-activating units (6 -10). Although intriguing, these studies utilize cellular overexpression systems where it is difficult to know the exact nature of protein complexes formed between the receptors.To study the function of isolated GPCRs, our laboratory and others have utilized a novel phospholipid bilayer reconstitution method (11-16). In this approach purified GPCRs are reconstituted into the phospholipid bilayer of a high density lipoprotein (HDL) particle. The reconstituted HDL (rHDL) particles are monodispersed, uniform in size, and preferentially incorporate a GPCR monomer (14, 15). Previous work in our lab has shown that rhodopsin, a class A GPCR previously proposed to function as a dimer (17)(18)(19), is fully capable of activating its G protein when reconstituted as a monomer in the rHDL lipid bilayer (15). Moreover, we have demonstrated that agonist binding to a monomeric ␤ 2 -adrenergic receptor, another class A GPCR, can be allosterically regulated by G proteins (14). This led us to determine whether a monomer of MOR, a class A GPCR that endogenously binds peptide ligands, is the minimal functional unit required to activate coupled G proteins. We ...

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Section: Discussionmentioning

confidence: 99%

Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors

Kuszak

Pitchiaya

Anand

et al. 2009

Journal of Biological Chemistry

162

155

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“…If we except harsh detergents such as SDS or N-lauroyl sarcosine (NLS), it is difficult to predict which detergent will be suitable for solubilization. Some methods such as immunoblot quantification [27,28] or fluorescence measurements using green fluorescent protein (GFP)-tagged receptors allow quantification of the total yield of solubilization [29][30][31][32]. In the latter experiments, two different GPCRs were expressed in the same host, Pichia pastoris.…”

Section: Quantification Of the Yield Of Solubilizationmentioning

confidence: 99%

“…Although DM was not the best solubilizing detergent, it was chosen because the authors argue that it was already used for successful crystallization of membrane proteins. In contrast, DM was unable to solubilize the m- [30]. Mirzabekov et al [33] used another method and tested a broad spectrum of detergent conditions to determine the detergent that allowed solubilization and isolation of native CCR5.…”

Section: Quantification Of the Yield Of Solubilizationmentioning

confidence: 99%

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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“…Les corps d'inclusion représentent une alternative intéressante, mais la protéine recombinante doit être manipulée en conditions dénaturantes, puis être ensuite repliée sous forme native et fonctionnelle. La production du récepteur NTS1 de la neurotensine est sans conteste le plus bel exemple de succès 1 [5]. La structure 3D du complexe récepteur H1 de l'histamine avec la doxepine (antagoniste) vient d'être publiée, suite à l'expression de ce RCPG chez P. pastoris [6].…”

Section: Expression Et Production Bactérienne Chez E Coliunclassified

Manipulation des récepteurs couplés aux protéines G

Banères

Mouillac

2012

Med Sci (Paris)

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> Parmi les différentes classes de protéines membranaires, les récepteurs couplés aux protéines G (RCPG) représentent une famille majeure. Ils sont impliqués dans la plupart des grandes fonctions physiologiques et jouent un rôle primordial dans la communication intercellulaire et la réception des signaux sensoriels. Ils constituent la cible de 30 % des médicaments actuellement sur le marché pharmaceutique. Pour comprendre le fonctionnement de ces récepteurs, aborder leur structure tridimensionnelle et déve-lopper des médicaments sélectifs et efficaces, il est nécessaire de purifier ces protéines afin de bien les caractériser. Cependant, cette étape n'est pas triviale, car les RCPG sont présents en très petite quantité dans la membrane plasmique, et leur environnement lipidique naturel suppose une extraction et une manipulation à l'aide de détergents. Il n'existe pas d'approche universelle aujourd'hui qui permette la production, la purification et la stabilisation des RCPG. Chacun de ces récepteurs possède des caractéristiques physicochimiques uniques, une préférence particulière pour certains détergents lors de leur solubilisation et des conditions spécifiques pour leur purification. Ces dernières années, de grandes avancées en termes de surexpression, de purification et surtout de stabilisation des RCPG, en particulier grâce aux amphipols et aux nanodisques, ont ouvert des perspectives très encourageantes pour l'étude structurale et l'analyse dynamique de ces protéines membranaires. Ces différents aspects de la manipulation des RCPG sont développés dans cette revue. < avec des analyses biochimiques et structurales. Leur surexpression est donc une condition préalable obligatoire si l'on veut aborder leur structure moléculaire, leur dynamique au cours de la liaison de ligands (drogues, médicaments) ou l'étude de leurs interactions avec des partenaires de signalisation tels que les protéines G ou les arrestines. Le développement de systèmes d'expression hétérologues capables de produire des protéines recombinantes fonctionnelles avec des rendements élevés constitue donc une étape essentielle. Une large palette de systèmes cellulaires adaptés à la production des RCPG est aujourd'hui disponible (voir quelques exemples représentatifs dans le Tableau I). Expression et production bactérienne chez E. coliLa bactérie Escherichia coli est le système de production de protéines membranaires le plus commun. Il est simple, peu coûteux, le temps de génération du bacille est court, il peut être manipulé sans danger, et les volumes de culture peuvent être aisément augmentés. De plus, les rendements d'expression sont élevés et la protéine recombinante est structuralement homogène (pas de modifications post-traductionnelles chez E. coli). Les RCPG sont ciblés soit à la membrane interne, soit en corps d'inclusion cytoplasmiques, dans les deux cas sous forme de protéines de fusion par un couplage à un partenaire ou un épitope court. Les corps d'inclusion représentent une alternative intéressante, mais la protéine recombinante doit ê...

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Solubilization, purification, and mass spectrometry analysis of the human mu-opioid receptor expressed in Pichia pastoris

Cited by 44 publications

References 33 publications

Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors

Purification and Functional Reconstitution of Monomeric μ-Opioid Receptors

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

Manipulation des récepteurs couplés aux protéines G

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