RESUMEN
G protein coupled receptors (GPCRs) function as guanine nucleotide exchange factors (GEFs) at heterotrimeric G proteins, and conduct this role embedded in a lipid bilayer. Detergents are widely used to solubilise GPCRs for structural and biophysical analysis, but are poor mimics of the lipid bilayer and may be deleterious to protein function. Amphipathic polymers have emerged as promising alternatives to detergents, which maintain a lipid environment around a membrane protein during purification. Of these polymers, the polymethacrylate (PMA) polymers have potential advantages over the most popular styrene maleic acid (SMA) polymer, but to date have not been applied to purification of membrane proteins. Here we use a class A GPCR, neurotensin receptor 1 (NTSR1), to explore detergent-free purification using PMA. By using an NTSR1-eGFP fusion protein expressed in Sf9 cells, a range of solubilisation conditions were screened, demonstrating the importance of solubilisation temperature, pH, NaCl concentration and the relative amounts of polymer and membrane sample. PMA-solubilised NTSR1 displayed compatibility with standard purification protocols and millimolar divalent cation concentrations. Moreover, the receptor in PMA discs showed stimulation of both Gq and Gi1 heterotrimers to an extent that was greater than that for the detergent-solubilised receptor. PMA therefore represents a viable alternative to SMA for membrane protein purification and has a potentially broad utility in studying GPCRs and other membrane proteins.
Asunto(s)
Ácidos Polimetacrílicos/química , Receptores de Neurotensina , Detergentes/química , Humanos , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/química , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Escherichia coli (E. coli) is the most widely used expression host for recombinant proteins due to high expression yields and straightforward molecular cloning. Directed evolution of G protein-coupled receptors (GPCRs) has made several of these difficult to express membrane proteins amenable to prokaryotic expression. Here, we describe a protocol for near complete 13CH3-methionine labeling of a thermostable neurotensin receptor 1 (enNTS1) variant in E. coli for solution NMR-based dynamics studies. Our expression strategy utilizes methionine biosynthesis pathway inhibition forcing E. coli to incorporate exogenous methionine with 96% efficiency at expression levels of 2.6 mg enNTS1 per liter of expression culture containing 50 mg of 13CH3-methionine. We also provide a 3-step purification protocol that produces final yields of 0.6 mg of functional Apo-state enNTS1.
Asunto(s)
Escherichia coli/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Metionina/química , Receptores de Neurotensina/aislamiento & purificación , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Isótopos de Carbono/química , Escherichia coli/genética , Humanos , Mutación , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
G protein coupled receptors (GPCRs) are central players in recognizing a variety of stimuli to mediate diverse cellular responses. This myriad of functions is accomplished by their modular interactions with downstream intracellular transducers, such as heterotrimeric G proteins and arrestins. Assembling a specific GPCR-G protein pair as a purified complex for their structural and functional investigations remains a challenging task, however, because of the low affinity of the interaction. Here, we optimized fusion constructs of the Gα subunit of the heterotrimeric G protein and engineered versions of rat Neurotensin receptor 1 (NTR1), coexpressed and assembled in vivo with Gß and Gγ. This was achieved by using the baculovirus-based MultiBac system. We thus generated a functional receptor-G protein fusion complex, which can be efficiently purified using ligand-based affinity chromatography on large scales. Additionally, we utilized a purification method based on a designed ankyrin repeat protein tightly binding to Green Fluorescent Protein (GFP-DARPin) that may be used as a generic approach for a large-scale purification of GPCR-G protein fusion complexes for which no ligands column can be generated. The purification methods described herein will support future studies that aim to understand the structural and functional framework of GPCR activation and signaling.
Asunto(s)
Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades gamma de la Proteína de Unión al GTP/aislamiento & purificación , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Repetición de Anquirina , Técnicas de Cultivo Celular por Lotes/métodos , Cromatografía de Afinidad/métodos , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Unión Proteica , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.
Asunto(s)
Cromatografía de Afinidad/métodos , Escherichia coli/metabolismo , Receptores de Neurotensina , Escherichia coli/genética , Humanos , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/química , Receptores de Neurotensina/genética , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
BACKGROUND: Purification of recombinant membrane receptors is commonly achieved by use of an affinity tag followed by an additional chromatography step if required. This second step may exploit specific receptor properties such as ligand binding. However, the effects of multiple purification steps on protein yield and integrity are often poorly documented. We have previously reported a robust two-step purification procedure for the recombinant rat neurotensin receptor NTS1 to give milligram quantities of functional receptor protein. First, histidine-tagged receptors are enriched by immobilized metal affinity chromatography using Ni-NTA resin. Second, remaining contaminants in the Ni-NTA column eluate are removed by use of a subsequent neurotensin column yielding pure NTS1. Whilst the neurotensin column eluate contained functional receptor protein, we observed in the neurotensin column flow-through misfolded NTS1. METHODS AND FINDINGS: To investigate the origin of the misfolded receptors, we estimated the amount of functional and misfolded NTS1 at each purification step by radio-ligand binding, densitometry of Coomassie stained SDS-gels, and protein content determination. First, we observed that correctly folded NTS1 suffers damage by exposure to detergent and various buffer compositions as seen by the loss of [(3)H]neurotensin binding over time. Second, exposure to the neurotensin affinity resin generated additional misfolded receptor protein. CONCLUSION: Our data point towards two ways by which misfolded NTS1 may be generated: Damage by exposure to buffer components and by close contact of the receptor to the neurotensin affinity resin. Because NTS1 in detergent solution is stabilized by neurotensin, we speculate that the occurrence of aggregated receptor after contact with the neurotensin resin is the consequence of perturbations in the detergent belt surrounding the NTS1 transmembrane core. Both effects reduce the yield of functional receptor protein.
Asunto(s)
Cromatografía de Afinidad/instrumentación , Receptores de Neurotensina/aislamiento & purificación , Resinas Sintéticas/química , Adsorción , Detergentes/química , Neurotensina/química , Estabilidad Proteica , Receptores de Neurotensina/química , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismoRESUMEN
Structural and functional analysis of most G-protein-coupled receptors (GPCRs) requires their expression and purification in functional form. The produced amount of recombinant membrane-inserted receptors depends on the optimal combination of a particular GPCR and production host; optimization of expression is still a matter of trial-and-error. Prior to purification, receptors must be extracted from the membranes by use of detergent(s). The choice of an appropriate detergent for solubilization and purification is crucial to maintain receptors in their functional state. The initial enrichment can be carried out by affinity chromatography using a general affinity tag (e.g., poly-histidine tag). If the first purification step does not yield pure receptor protein, purification to homogeneity can often be achieved by use of a subsequent receptor-specific ligand column. If suitable immobilized ligands are not available, size exclusion chromatography or other techniques need to be applied. Many GPCRs become unstable upon detergent extraction from lipid membranes, and measures for stabilization are discussed. As an example, the purification of a functional neurotensin receptor to homogeneity in milligram quantities is given below.
Asunto(s)
Receptores Acoplados a Proteínas G/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Animales , Cromatografía de Afinidad/métodos , Humanos , Receptores Acoplados a Proteínas G/química , Receptores de Neurotensina/química , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes/química , SolubilidadRESUMEN
The G-protein coupled receptor (GPCR), rat brain neurotensin receptor type I (NTS1) is one of a small number of GPCRs that have been successfully expressed in Escherichia coli as a functional, ligand-binding receptor, but yields of purified receptor are still low for comprehensive structural studies. Here, several approaches have been examined to optimize the yields of active, ligand-binding receptor. Optimisation of E. coli strain and induction protocol yielded a significant improvement in expression of active receptor. Expression of the receptor in BL21(DE3) cells, in combination with autoinduction improved expression 10-fold when compared with previously reported expression protocols using IPTG-mediated induction in DH5alpha cells. Optimization of the purification protocol revealed that supplementation of buffers with phospholipids enhanced recovery of active receptor. The methods examined are potentially applicable to other GPCRs expressed in E. coli.
Asunto(s)
Receptores Acoplados a Proteínas G/metabolismo , Receptores de Neurotensina/genética , Secuencia de Aminoácidos , Animales , Tampones (Química) , Escherichia coli/genética , Concentración de Iones de Hidrógeno , Ligandos , Datos de Secuencia Molecular , Neurotensina/aislamiento & purificación , Neurotensina/metabolismo , Fosfolípidos/química , Unión Proteica/genética , Ratas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/química , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , TemperaturaRESUMEN
NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.
Asunto(s)
Receptores de Neurotensina/química , Receptores de Neurotensina/genética , Animales , Fenómenos Biofísicos , Biofisica , Escherichia coli , Humanos , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/aislamiento & purificaciónRESUMEN
G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of approximately 1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of approximately 2. NTS1 monomers activate G alpha q beta(1)gamma(2), whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Receptores de Neurotensina/metabolismo , Unión Competitiva/efectos de la radiación , Cromatografía en Gel , Dimerización , Humanos , Luz , Peso Molecular , Neurotensina/metabolismo , Unión Proteica/efectos de la radiación , Receptores de Neurotensina/aislamiento & purificación , Refractometría , Dispersión de Radiación , Relación Estructura-Actividad , Rayos UltravioletaRESUMEN
Structure determination of G-protein-coupled receptors and other applications, such as nuclear magnetic resonance (NMR) studies, require milligram quantities of purified, functional receptor protein on a regular basis. This unit presents a step-by-step procedure for the automated two-column purification at the 10-milligram scale of a G protein-coupled receptor for neurotensin, expressed in functional form in Escherichia coli.
Asunto(s)
Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores de Neurotensina/aislamiento & purificación , Automatización , Cromatografía de Afinidad , Proteínas Recombinantes/aislamiento & purificación , SolubilidadRESUMEN
Structure determination of G-protein-coupled receptors and other applications, such as nuclear magnetic resonance studies, require milligram quantities of purified, functional receptor protein on a regular basis. We present an overview on expression and purification studies with a receptor for neurotensin. Functional expression in Escherichia coli and an automated two-column purification routine allow ongoing crystallization experiments and studies on receptor-bound ligands.
Asunto(s)
Conformación Proteica , Proteómica/métodos , Receptores de Neurotensina/aislamiento & purificación , Receptores de Neurotensina/metabolismo , Cristalización/métodos , Electroforesis en Gel de Poliacrilamida , Escherichia coliRESUMEN
In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.
Asunto(s)
Receptores Acoplados a Proteínas G/biosíntesis , Receptores Acoplados a Proteínas G/genética , Animales , Sistema Libre de Células , ADN/genética , Detergentes , Digitonina , Escherichia coli/genética , Escherichia coli/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/genética , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/aislamiento & purificación , Subunidades alfa de la Proteína de Unión al GTP Gs/biosíntesis , Subunidades alfa de la Proteína de Unión al GTP Gs/genética , Subunidades alfa de la Proteína de Unión al GTP Gs/aislamiento & purificación , Vectores Genéticos , Humanos , Técnicas In Vitro , Cinética , Fusión de Membrana , Polidocanol , Polietilenglicoles , Biosíntesis de Proteínas , Ratas , Receptor Muscarínico M2/biosíntesis , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/aislamiento & purificación , Receptores Adrenérgicos beta 2/biosíntesis , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/aislamiento & purificación , Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores de Neurotensina/biosíntesis , Receptores de Neurotensina/genética , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Solubilidad , Tiorredoxinas/biosíntesis , Tiorredoxinas/genética , Tiorredoxinas/aislamiento & purificaciónRESUMEN
Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.
Asunto(s)
Neurotensina/metabolismo , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Cromatografía de Afinidad , Endopeptidasas/metabolismo , Fermentación , Receptores de Neurotensina/genética , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
G-protein-coupled receptors (GPCRs) are integral membrane proteins involved in signal transduction and constitute major drug targets for disease therapy. Aptamers, which are globular RNA or DNA molecules evolved to specifically bind a target, could represent a valuable tool with which to probe the role of such receptors in normal tissue and disease pathology and for cocrystallization with receptors for structure determination by X-ray crystallography. Using the bacterially expressed rat neurotensin receptor NTS-1 as an example, we describe a strategy for the generation of GPCR-specific RNA aptamers. Seven rounds of a "subtractive," paramagnetic bead-based selection protocol were used to enrich for neurotensin receptor-specific aptamers, while circumventing the evolution of aptamers reactive to minor protein contaminants. Representatives of each aptamer family were analyzed in Escherichia coli membrane nitrocellulose filter binding assays. Eight aptamers demonstrated specificity for the neurotensin receptor. One aptamer, P19, was characterized in detail and shown to bind to both the rat receptor and the human receptor with nanomolar affinity. P19 was also shown to interact with rat neurotensin receptor expressed in CHO cells, in both membrane preparations and intact cells. P19 represents the first example of a GPCR-specific RNA aptamer.
Asunto(s)
ARN/metabolismo , Receptores de Neurotensina/metabolismo , Animales , Proteínas de Unión al GTP/metabolismo , Neurotensina/metabolismo , Ratas , Receptores de Neurotensina/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ARN , TemperaturaAsunto(s)
Escherichia coli/genética , Receptores de Neurotensina/genética , Receptores de Neurotensina/aislamiento & purificación , Marcadores de Afinidad , Animales , Cromatografía de Afinidad , Técnicas In Vitro , Métodos , Neurotensina/metabolismo , Ratas , Receptores de Neurotensina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Immobilized metal affinity chromatography has recently been used for purification of histidine-tagged membrane proteins in the presence of detergents with varying success. Strong binding to the metal resin is essential for purification when expression levels are low. We have investigated the influence of tag length and type of detergent on the purification of a neurotensin receptor fusion protein expressed in Escherichia coli at a level of about 0.1% of membrane protein. Receptors with six C-terminal histidine residues did not bind to nickel resin in the presence of the anionic detergent sodium dodecyl sulfate. In contrast, partial purification assessed by densitometry of Coomassie-stained gels was achieved using the nonionic detergents dodecyl maltoside or Triton X-100 (53% pure), or a detergent mixture containing the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (46% pure). Linking a highly charged epitope tag to the histidine tail did not affect the nickel-binding properties of receptors. The level of purification was substantially improved (72% pure) by extending the histidine tail to 10 residues because this allowed stringent washes at high imidazole concentration to remove nonspecifically bound contaminants. This strategy not only resulted in efficient purification of receptors from crude membranes, but also worked particularly well for single-step purification from total cell lysates, resulting in 340-fold purification of functional neurotensin receptor.
Asunto(s)
Cromatografía de Afinidad/métodos , Receptores de Neurotensina/aislamiento & purificación , Detergentes , Epítopos , Escherichia coli , Níquel , Oligopéptidos , Péptidos , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusión/aislamiento & purificaciónRESUMEN
A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress.
Asunto(s)
Receptores de Neurotensina/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Cromatografía de Afinidad , Clonación Molecular , ADN Complementario , Detergentes , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Lectinas de Unión a Manosa , Datos de Secuencia Molecular , Ratas , Receptores de Neurotensina/genética , Proteínas Recombinantes de Fusión/genética , SolubilidadRESUMEN
High affinity neurotensin receptors were solubilized in an active form from newborn human brain using the non-denaturing detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The solubilized receptor was purified in a single step by affinity chromatography. The binding properties of the purified receptor towards [125I-Tyr3]neurotensin are very similar to those of the membrane bound and of the crude CHAPS-solubilized receptor in terms of affinity and specificity. The purified receptor is a single protein chain of molecular weight 100 kDa as shown by gel filtration and by affinity labelling with [125I-Tyr3]neurotensin in the presence of the cross-linking agent disuccinimidyl suberate.