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Protein Expression and Purification 55 (2007) 300–311

High-level expression and purification of the human bradykininB2 receptor in a tetracycline-inducible stable HEK293S cell line

Pamela Camponova 1, Stephanie Baud 1, Helene Mattras 1, Isabelle Duroux-Richard 1,Jean-Claude Bonnafous 1, Jacky Marie *,1

Centre de Biochimie Structurale, UMR CNRS 5048, INSERM 554, 29 rue de Navacelles, 34090 Montpellier Cedex, France

Received 15 March 2007, and in revised form 13 April 2007Available online 5 May 2007

Abstract

The B2 bradykinin receptor belongs to the G-protein coupled receptor family. Development of new drugs for this important thera-peutic target requires structural information on the receptor. The main goal of the present work was to overexpress the human B2 recep-tor for future biophysical studies. Different tagged B2 receptors were engineered and their properties were evaluated by transientexpression in HEK293S cells. A B2 receptor tagged with a hexahistidine at the N-terminus and a nonapeptide at the C-terminus wasselected for high expression level and preserved ligand-binding characteristics. First, we generated a HEK293S stable cell line expressingthe receptor constitutively at a level of 60 pmol/mg of crude membrane protein. However, the decrease of expression level with cell pas-sages led us to express the B2 receptor in a HEK293S tetracycline-inducible stable cell line. Induction of expression of the B2 receptorwith tetracycline and sodium butyrate led to a level of 100 pmol/mg of membrane protein, which is the highest level reported so far forthis receptor. The expression level was stable with cell passages and the ligand-binding and signal transduction properties of the receptorwere unaltered. The receptor was purified to near homogeneity by solubilization with n-dodecyl-b-D-maltoside followed by a two-steppurification procedure combining hydroxyapatite and immunoaffinity chromatography. Although the purified receptor is not functional,the purification of the B2 receptor to near homogeneity from a stable cell line overexpressing this receptor pave the way for future struc-tural studies of this receptor.� 2007 Elsevier Inc. All rights reserved.

Keywords: G-protein coupled receptor; Bradykinin; B2 receptor; Mammalian cells; HEK293S; Overexpression; Purification

G-protein coupled receptors (GPCRs)2 are integralmembrane proteins composed of seven transmembrane

1046-5928/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.pep.2007.04.020

* Corresponding author. Present address: Institut des Biomolecules MaxMousseron, UMR 5247 CNRS-UM1-UM2. Faculte de Pharmacie, 15avenue Charles Flahaut, Bat. K, BP 14491, 34093 Montpellier Cedex 5,France. Fax: +33 4 67 54 86 25.

E-mail address: jacky.marie@univ-montp1.fr (J. Marie).1 Fax: +33 4 67 41 79 13.2 Abbreviations used: GPCR, G-protein coupled receptor; BK, brady-

kinin; B2, bradykinin B2 subtype; HEK, human embryonic kidney; HPP-HOE-140, hydroxyphenyl-propionyl-HOE-140; DM, n-dodecyl-b-D-mal-toside; TetR, tet operon repressor protein; TetO, tet operator; FBS, fetalbovine serum; NHS, N-hydroxysuccinimide; RuðbipyÞ2þ3 , ruthenium (II)tris-bipyridyl dication; APS, ammonium persulfate; PBS, phosphate-buffered saline; IP, inositol phosphate; NTA, nitrilotriacetic acid.

spanning domains [1,2]. In human genome more than 800GPCRs have been identified and classified in five mainfamilies [3]. GPCRs recognize a wide variety of ligands(photons, ions, peptides, amines, proteins, lipids, photons,odors and gustative molecules) [2]. GPCRs are involved inthe main physiological processes and it is estimate that halfof the currently available drugs target these receptors [4,5].To date, only 10% of the GPCRs are targeted, making drugdiscovery of these receptors a tremendous potential marketfor pharmaceutical companies [6]. Compelling informa-tions regarding the structure of ligand–receptor complexeswill facilitate the design of new specific drugs. However, theonly one structure of a ligand–receptor complex availableat high resolution is that of bovine rhodopsin isolated frombovine retinas [7,8].

P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311 301

The scarce structural information for GPCRs comesfrom the difficulties associated with obtaining largeamounts of protein in a pure active form and stable forstructural studies [9,10]. The first limitation is that mostGPCR are expressed at low levels in their natural environ-ment. Therefore, overexpression of the receptor in a heter-ologous cell system is required with the exception ofrhodopsin which is expressed at very high levels in rod cellsof retinae. Once high-level recombinant expression hasbeen achieved, one of the two strategies must be followed[11]. The first is to overexpress the protein in an active format the plasma membrane of eukaryotes cells or at the innermembrane of Escherichia coli and then to preserve theactivity of the protein during the solubilization and purifi-cation steps. The second strategy requires overexpressionof the protein in a unfolded inactive state, such as in inclu-sion bodies in E. coli and then to solubilize, purify and thenrefold the protein to produce a functional form.

The limitation of the first strategy lies in difficultyobtaining the recombinant protein at very high levels atthe cell plasma membrane. For the second strategy, it isoften difficult to find conditions for efficient refolding. Suc-cess has been reported for the two strategies. To date, how-ever, poor understanding of protein folding mechanismsand translocation to the plasma membrane still limit devel-opment of an optimal cell system applicable to overexpres-sion of all GPCRs. Thus, overexpression of eukaryoticmembrane proteins requires testing of the different expres-sion cells systems and requires optimization of purificationschemes or finding conditions for refolding [11].

This study focuses on overexpression of the human bra-dykinin (BK) B2 receptor which belongs to family A ofGPCRs (the rhodopsin family) [3]. Bradykinin B2 receptorand its subtype bradykinin B1 receptor are receptors forkinins (BK, Lys-BK bind the B2 receptor and des-Arg9-BK, Lys-Des-Arg9-Bk bind the B1 receptor) [12]. The B2

receptor plays crucial roles in human pathophysiology thatmakes this receptor a potential target for treatment of car-diovascular disorders, pain and inflammation [13].

In this study, we describe the overexpression of the bra-dykinin B2 receptor in a mammalian cell for future struc-tural studies. Two type of stable HEK293S cell linesexpressing an affinity tagged receptor have been developed;one expressing the receptor constitutively, the secondexpressing the receptor under a tetracycline-inducible pro-moter. Binding and coupling characteristics of theexpressed receptors were analyzed and a purification proto-col for the receptor from the tetracycline induced cell linewas designed.

Materials and methods

Materials

BK was purchased from Sigma, myo-[2-3H] inositol(16 Ci/mmol) and (prolyl 2,4-3,4-3H) bradykinin([3H]BK) (40–100 Ci/mmol) were purchased from Amer-

sham Pharmacia Biotech. Hydroxyphenyl-propionyl-HOE-140 (HPP-HOE-140) was kindly supplied by Profes-sor J. Martinez (CNRS, Montpellier, France); it wasradioiodinated using [125I] Na (2000 Ci/mmol) andIODO-GEN as oxidizing agent. HEK293S and HEK293STetR cells expressing the Tet operon repressor protein werekindly provided by P.J. Reeves (present address: Universityof Essex, Colchester, UK). The detergent n-dodecyl-b-D-maltoside (DM) was purchased from Anatrace. The rho-1D4 antibody specific to the C9 epitope was purchasedfrom the University of British Columbia, the nonapeptide(C9) corresponding to the C terminal sequence of rhodop-sin [14] was generously synthesized by Alain Chavanieuand Jean-Francois Guichou (Centre de Biochimie Structu-rale, CNRS 5048, INSERM 554, Montpellier, France).Blasticidin, penicilin–streptomycin solutions were fromInvitrogen. Geneticin, Trypsin–EDTA, glutamine DMEM,DMEM/F12 (1/1) were from GIBCO/BRL. Poly-L-orni-thine (Mr 30.000–70.000) was from Sigma, FBS was fromHyclone, NHS-activated Sepharose was from Amersham.

Methods

Generation of tagged B2 receptor constructs

Four constructs containing c-Myc, hexahistidine or C9tags at the N- and/or C-terminus were created.

(A) c-Myc at the C-terminus, (B) c-Myc at the N-termi-nus and C9 at the C-terminus, (C) c-Myc at the N-terminusand His at the C-terminus, (D) His at the N-terminus andC9 at the C-terminus.

All constructs were generated by PCR using the B2

receptor cDNA cloned in the PRK5 vector as a templateand Pfu polymerase (Stratagene). The forward oligonucle-otides contained an EcoRI site followed by a Kozaksequence just upstream of the ATG start codon and thereverse oligonucleotide contained an XbaI site just down-stream of the TGA stop codon. All constructs were firstcloned in the PRK5 vector (Pharmingen) by digesting boththe PCR products and PRK5 vector with EcoRI and XbaIenzymes. The constructs in the PRK5 vector were verifiedby DNA sequence analysis.

The construct D was then cloned in the pACHEnc forexpression in stable HEK293S cell line. The pACHEnc vec-tor, a derivative of the vector described by Velan et al. [15],was provided by Dr. A. Shafferman (Israel Institute forBiological Research, Ness-Ziona, Israel). The PRK5 vectorcontaining the construct D was digested by EcoRI, the sitewas then filled using the Klenow fragment of polymerase Iand dNTPs and the insert was excised from PRK5 withSalI. The insert was the cloned in the pACH vectordigested with EcoRV and SalI.

For generation of inducible stable cell line, the constructD was cloned in the pACMV-TetO vector kindly providedby P.J. Reeves (University of Essex, Colchester, UK) [16].To do this, the insert was excised from the PRK5 vectorwith EcoRI and SalI enzymes and cloned first into the Eco-RI and SalI sites of the pIRES-neo vector (Clonetech).

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pIRES-neo was only used as an intermediary vector inorder to insert the restriction sites required for cloning inthe pACMV-TetO vector. Then, the insert was excisedfrom the pIRES-neo vector by digestion with NheI andNotI and cloned in the NheI and NotI sites of the pAC-MV-TetO vector.

Construction of the stable HEK293S cell line for constitutive

B2 receptor expression

HEK293S cells were maintained in DMEM/F12 (1/1)medium supplemented with 10% FBS, penicillin (100 U/ml), streptomycin (100 lg/ml) and glutamine (2 mM).Plastic dishes used for HEK293S cells culture were system-atically coated with Poly-L-ornithine. For constitutivereceptor expression, the cells were transfected with thepACH vector containing the RB2 cDNA constructs usingthe calcium phosphate precipitation method as followed.Briefly cells in exponential phase were seeded at a densityof 1–2 · 106 cells per 10 cm plate in 10 ml DMEM supple-mented with 10% FBS, antibiotic and glutamine as forDMEM/F12 medium. Twenty-four hours later, cells weretreated with the plasmid (30 lg) mixed with 500 ll ofCaCl2 (0.25 M) and 500 ll of BES buffer (50 mM BES,1.5 mM Na2HPO4, pH 7.02, 250 mM NaCl) and incu-bated in a humidified incubator under 5% CO2, at 37 �Cfor 24 h. Cells were then trypsinized and seeded at a den-sity of 1 · 106 cells per 10 cm plate and incubated for afurther 24 h. The medium was then replaced by fresh med-ium containing 800 U/ml geneticin and was changed every2–3 days. 15–20 days later, colonies are isolated using pip-ette tips and then transferred in 24-well dishes andexpanded through 12-well, 6-well and finally 10 cm plates.At confluence, the cells are trypsinized and seeded in a10 cm plate for freezing in liquid nitrogen and in 48-welldishes for detection of receptor expression by binding testswith [3H]BK. Clones with the highest expression level ofreceptors was then selected further with 1.2 mg/mlgeneticin.

Construction of the stable HEK293S-TetO cell line for

tetracycline induced B2 receptor

HEK293S cell line stably transfected with the Tetoperon repressor protein (HEK293S TetR) was kindlyprovided by P.J. Reeves [16]. The HEK293S TetR cell linewas maintained under blasticidin selection in DMEM/F12(1/1) medium containing blasticidin (5 lg/ml) and supple-mented with 10% FBS, penicillin (100 U/ml), streptomy-cin (100 lg/ml) and glutamine (2 mM). HEK293S TetRcells were transfected with the pACMV-TetO-RB2 by cal-cium phosphate precipitation method. Briefly cells inexponential phase were seeded at a density of 1–2 · 106

cells per 10 cm plate in 10 ml DMEM supplemented with10% FBS, antibiotic and glutamine as for DMEM/F12medium.

Twenty-five hours later, cells were treated with the plas-mid (30 lg) mixed with 500 ll of CaCl2 (0.25 M) and 500 llof BES buffer (50 mM BES, 1.5 mM Na2HPO4, pH 7.02,

250 mM NaCl) and incubated in a humidified incubatorunder 5% CO2, at 35 �C for 19 h. Clones were selected withDMEM/F12 medium containing geneticin as described forthe constitutive cell line, except that the geneticin concen-tration was maintained at 1 mg/ml during the selectionprocedure.

Transient receptor expression

Receptors were transiently expressed in HEK293S cellsusing the electroporation transfection method as alreadydescribed [17]. Briefly, 107 cells were incubated for 10 minat room temperature with 20 lg PRK5 vector and variousquantities of different constructs (0.1–1lg) in 300 ll of elec-troporation buffer (50 mM K2HPO4, 20 mM KOH, 20 mMCH3COOK and 27 mM MgSO4, pH 7.4) in an electropor-ation cuvette (0.4 cm, Bio-Rad). The solution was submit-ted to an electric discharge (950 lF, 270 V) andimmediately transferred to fresh culture medium(DMEM/F12 medium, 10% fetal calf serum, 100 U/mlpenicillin, 100 U/ml streptomycin). Cells were culturedtwo days before performing the pharmacological tests.

Binding experiments

Crude plasma membranes. Crude membranes fromHEK293S were prepared as described previously [18].[3H]BK-binding assays were performed as follows: themembranes were resuspended in binding buffer [25 mMN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid,140 lg/ml bacitracin, 1 mg/ml bovine serum albumin,1 mM o-phenanthroline, pH set to 6.8 with ammonia]and incubated for 1.5 h at 25 �C in binding buffer in thepresence of [3H]BK using a 6 · 10�11 to 10�8 M range.Nonspecific binding was evaluated in the presence of10�6 M unlabeled BK. Each point was done in triplicate.Bound radioactivity was separated from free ligand by fil-tration through GF/C filters presoaked in 0.1% polyethyl-eneimine. Radioactivity was measured by liquidscintillation counting. Dissociation constant (Kd) and max-imum expression level (Bmax) were determined from Scat-chard plots.

Intact cells. Binding to transiently transfected HEK293Scells or HEK293S cell lines grown in 48- or 24-well tissueculture clusters (about 1 · 105 to 2 · 105 cells/well, respec-tively) was carried out at 4 �C in cell-binding buffer (Dul-becco’s phosphate-buffered saline supplemented with140 lg/ml bacitracin, 1 mg/ml bovine serum albumin,1 mM o-phenanthroline, and 105 M captopril, pH 7.0)using a 4 h incubation time, under gentle agitation. Totalbinding was estimated by using a concentration range0.15–10 nM of [3H]BK or [125I]HPP-HOE-140. Nonspecificbinding was determined using an excess of the correspond-ing unlabeled ligand (10�6 M). Each point was done in trip-licate. Bound radioactivity was evaluated after washing thecells twice with cold-binding medium and collecting themin 500 ll of 0.1 N NaOH. Radioactivity was measured byliquid scintillation counting.

P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311 303

Crosslinking of B2 receptor with [125I]HPP-HOE-140

The B2 receptor was photolabeled with the radioiodin-ated B2 receptor antagonist [125I]HPP-HOE-140 on intactcells by ruthenium chelate photosensitization as alreadydescribed [19]. Briefly, 107 cells in a 15 cm dish were incu-bated with [125I] HPP-HOE-140 (1 nM) in 30 ml of cell-binding buffer for 3 h at 4 �C under agitation. The bindingsolution was discarded and the cells were washed twicewith cold phosphate-buffered saline containing Ca2+ andMg2+. (PBS). Washed cells were incubated with 2 mMRuðbipyÞ2þ3 , 1 mM APS in 5 ml cold PBS and immediatelyirradiated for 3 s at 0 �C, by visible light (two 100 W tung-sten lamps) located 15 cm from the sample.

Inositol phosphate assay

HEK293S cell lines were grown to 60% confluence in12-well tissue culture clusters and labeled for 24 h withmyo-[2-3H]inositol (1 ml/well, 1 lCi/ml) in medium 199deprived of serum. Before stimulation, cells were incu-bated at 37 �C for 1 h in inositol phosphates (IP) buffer(consisting of 116 mM NaCl, 4.7 mM KCl, 2.5 mMCaCl2, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulf-onic acid, 11 mM glucose, 140 lg/ml bacitracin, and105 M captopril, pH 7.4). After a 15-min preincubationin IP buffer containing 10 mM LiCl, cells were incubatedin the presence or absence of ligands for 15 min at 37 �Cin medium containing 10 mM LiCl. Pooled IPs wereextracted and measured as described previously [17,20].

Induction of expression, solubilization and purification of theHis-C9 tagged B2 receptor

Induction of receptor expression. Routinely, induction wasdone on cells grown in a 15 cm plate. HEK293S TetO cellsexpressing the c-6His-RB2-C9 receptor were seeded at 2–3 · 106 cells per 15 cm plates. Twenty-four hours later,the cells were supplied with the induction medium (20 mlof complete DMEM/F12 medium) containing tetracycline(1–2 lg/ml) and/or sodium butyrate (5 mM) without genet-icin and blasticidin. The medium was replaced by freshinduction medium every 24 h. The induction conditionswere first optimized using cells grown in 48-well plates(5 · 104 cell/well).

Preparation of crude membranes. Cells grown on plateswere collected 48 h after incubation in medium containingtetracycline and sodium butyrate.

The cells were rinsed twice with 10 ml of cold PBS for a15 cm plate. The cells were collected by scrapping in 10 mlof 10 mM Tris–HCl, pH 7.4, 1 mM PMSF, then, brokenon ice using an Ultraturax polytron (three strokes 10 seach). The cell homogenate was centrifuged at 4 �C,5 min at 100g. The supernatant was collected and crudemembranes were pelleted by centrifugation at 39,000g for30 min at 4 �C. The pellet was resuspended at a proteinconcentration of 7–10 mg/ml in TES buffer (25 mM TES,pH 6.8, with complete protease inhibitor cocktail (Sigma)),aliquoted and stored in liquid nitrogen.

Receptor solubilization. Frozen membrane preparations(70 mg protein) were slowly thawed in ice and resus-pended with a Dounce homogenizer at a concentrationof 2.5 mg protein/ml in phosphate buffer (137 mMNaCl, 2.7 mM KCl, 1.5 mM K2HPO4, 8 mMNa2HPO4, pH 7.2) containing 1% (wt/vol) DM. Thehomogenate was end-over-end mixed for 45 min atroom temperature followed by a 45 min end-over-endmixing at 4 �C. The solubilized extract was centrifugedat 40,000 rpm in a Ti-50.2 rotor for 45 min at 4 �C andthe soluble fraction was collected and immediately usedfor purification.

Purification. The solubilized membrane extract (35 ml)was mixed with 4 ml of hydroxyapatite (HA) (Biogel-HTP, Bio-Rad) equilibrated in HA buffer (10 mM phos-phate, pH 7.0, 0.5% DDM) and incubated in batch for1 h at room temperature. The gel was then packed intoa column (15 mm width) and washed with six volumesof HA buffer. Adsorbed proteins were eluted with 4.5 mlof 0.3 M phosphate, pH 7.0, 0.5% DM. The eluate wasthen subjected to immunoaffinity chromatography usingrho-1D4-Sepharose gel. The rho-1D4 antibody was cou-pled on NHS-activated Sepharose 4 Fast Flow at a con-centration of 2.5 mg antibody/ml of gel by following theprocedure recommended by the manufacturer. The HAeluate was incubated overnight at 4 �C with 850 ll of1D4-Sepharose gel (1 ml) equilibrated in phosphate buffer,0.5% DM. The gel was then packed in a column (10 mmwidth) and washed sequentially at room temperature with20 volumes of 10 mM phosphate buffer, pH 7.0, DM0.5% and 10 volumes of citrate buffer (10 mMNa2HPO4/5 mM citric acid, pH 6.0) containing 0.2%DM. The receptor was eluted using citrate buffer, pH6.0, 0.2% DM containing 200 lM peptide C9. The eluate(1 ml) was concentrated to 200 ll with a 30 kDa MW cut-off Centricon ultrafiltration device (1500g for 30 min),washed with 1 ml of 10 mM phosphate buffer, pH 7.0,0.1% DM and concentrated again to a final volume of190 ll. The concentrated solution was incubated in batch30 min at room temperature with 30 ll of Ni–NTA aga-rose gel equilibrated with 10 mM phosphate buffer, pH7.0, 500 mM NaCl, 3 mM imidazole, 0.1% DM. The gelwas washed first with 600 ll of 10 mM phosphate, pH7.0, 0.1% DM, 20 mM imidazole followed with 600 ll ofthe same buffer containing 50 mM imidazole. The recep-tor was eluted with 120 ll of 10 mM phosphate, 0.1%DM, 200 mM imidazole.

N-Glycosidase F treatment

In some experiments, the receptor was deglycosylatedbefore Ni–NTA purification. The immunopurified receptorwas equilibrated by several round of concentration with aCentricon P-30 in 10 mM phosphate buffer, pH 8.2, 0.1%DM. 80 ll of the concentrated fraction corresponding to120 lg protein was incubated with 4 U of N-glycosidaseF (Roche) at 32 �C for 18 h.

Fig. 1. Schematic representation of the B2 bradykinin receptor constructs.(a) Diagram of the secondary structure of the human B2 receptor showingthe positions of tags at the N-terminus ( ) and C-terminus ( ). Thedouble arrow symbolizes the deletion of the three first aminoacids at Asn3

bearing potential glycan chains ( ). (b) Tags used in this study: c-Mycdecapeptide, hexa histidine, C9 nonapeptide. (v) Different tagged receptorconstructs used: c-Myc at the N-terminus (RB2 c-MycN), c-Myc at the N-terminus and 6His at the C-terminus (RB2 c-MycN-6His C), c-Myc at theN-terminus and C9 at the C-terminus (RB2 c-MycN-C9C) and 6His at theN-terminus and C9 at the C-terminus (RB26His N-C9 C).

Table 1Binding characteristics of wild-type and tagged B2 bradykinine receptors

Constructs [3H]BK binding

Kd (nM) Bmax (pmol/mg protein)

WT 0.27 ± 0.12 4.8 ± 1.5RB2-c-MycN 0.31 ± 0.20 5.4 ± 1.0RB2-c-MycN-6His C 0.51 ± 0.10 5.7 ± 0.8RB2-c-MycN-C9C 0.72 ± 0.30 10 ± 2.0RB2-6His N-C9C 0.55 ± 0.15 8.1 ± 1.5

The affinity constant (Kd) and maximal binding capacity (Bmax) weredetermined by Scatchard analysis of saturation binding experiments with[3H]BK on crude membranes prepared from HEK293S cells expressingtransiently the receptors. The cDNA amount for each receptor used fortransfection was the same (1 lg). Results are the means ± SEM of threeindependent experiments performed in triplicate.

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SDS–PAGE and western blotting

Proteins were separated by 10% SDS–PAGE underreducing conditions and then visualized by silver stain.For western blots, proteins from SDS–PAGE gel weretransferred to PVDF membranes (Bio-Rad) at 100 Vfor 1 h. PVDF membranes were then incubated inblocking buffer (20 mM Tris–HCl, pH 7.6, 136 mMNaCl, 10% defatted milk, 0.1% Tween 20) for 1 h atroom temperature. Blocked membranes were incubatedwith 30 lg of rho-1D4 antibody in blocking buffer for1 h at room temperature. After washing three times withTBS/Tween buffer (20 mM Tris–HCl, pH 7.6, 136 mMNaCl, 0.1% Tween 20), the membranes were incubatedwith goat anti-mouse IgG horseradish peroxidase anti-body (Jackson Immunoresearch) at a dilution of 1/4000 in blocking buffer for 1 h at room temperature.After washing three times with TBS/Tween buffer, theproteins were visualized with SuperSignal West PicoChemiluminescent Substrate (Pierce) using BioMax MRfilm (Kodak).

Autoradiography of iodinated samples was done byexposing dried gel or blotted membrane to BioMax XARfilm (Kodak). Quantification of silver stained proteinswas performed by densitometry scanning, using PC BAS2.09 program.

Protein estimation

The amount of protein was quantified by Bradfordmethod [21], or BCA protein assay (Pierce) when samplescontained detergent.

Results

Ligand-binding properties of tagged B2 receptor constructs

In order to evaluate the influence of the tags on ligandrecognition and expression level of the receptor, the differ-ent tagged receptor constructs were transiently expressed inHEK293S. As schematically represented in Fig. 1a, tagswere inserted at the N- and/or C-terminus of the B2 recep-tor truncated at the Asn3 glycosylation site. Indeed, sup-pression of the potential glycosylation site might favortag recognition and had no significant influence on the pha-ramocological properties of the receptors [17]. Three tagswere used (c-Myc, 6His, C9) (Fig. 1b). These tags wereadded in different combinations in order to engineer fourreceptor constructs (Fig. 1c). As indicated in Table 1, theaddition of tags did not changed significantly the affinityof [3H]BK for the different receptor constructs comparedto the WT receptor. Interestingly, we observed that thetag could have a beneficial effect on the expression of thereceptor at the surface of the cell (measured by bindingon cell membranes, by using [3H]BK as ligand). Thus, Bmax

values of receptors tagged with the C9 epitope at the C-ter-minus and c-Myc or 6His at the N-terminus (RB2-c-MycN-C9C and RB2-6His N-C9C) were 2-fold that of the WTreceptor (Table 1).

We decided to use RB2-6His N-C9C for expression instable cell line because preliminary experiments showedthat 6His tag might be more efficient than c-Myc tag forpurification on Ni–NTA in combination with immunoaf-finity purification with anti-C9 rho-1D4 antibody.

P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311 305

Constitutive expression of RB2-6His N-C9C in stable

HEK293S cell line

Initially we generated a HEK293S cell line expressingthe construct RB2-6His N-C9C cloned into the pACHvector. Different clones were tested for expression of thereceptor at the surface of the cell. Saturation-bindingexperiments performed on crude plasma membranes using[3H]BK showed that the binding was saturable with only asingle class of binding sites present. The Kd of [3H]BK forthe expressed receptor was 0.4 nM (Fig. 2). This affinityconstant is in good agreement with that of the wild typeB2 receptor expressed in others transfected mammaliancells [22,23] and in native human cells [24].

The maximum expression level obtained was 60 pmol/mg of crude membrane proteins for clones selected withhigh concentration of geneticin (1 mg/ml). When testedin intact cells the expression level corresponded to3 · 106 sites/cell. This expression level is one of the highestreported for expression of bradykinin B2 receptor com-pared with other cell systems (see Discussion). Unfortu-nately, we observed that the level of expression graduallydecreased with cell passage number and stabilized in therange of 20–30 pmol/mg protein after 8–10 passages.Because this effect might have arisen from a loss of copiesof inserted pACH vector itself or a toxic effect resultingfrom overexpression of the receptor, we decided to createan inducible stable cell line in order to limit this phenome-non and to ensure more reproducible expression levels.

Expression of RB2-6His N-C9C in a inducible stableHEK293S cell line

The transfected HEK293S cells were selected with 1 mg/ml geneticin. Expanded colonies were then tested individu-ally for binding using a saturating concentration of[3H]BK. Cells were grown at near confluence in 48-well

Fig. 2. Binding characteristics of the RB2-6His N-C9C receptor expressedconstitutively in the HEK293S stable cell line. Scatchard representation ofa saturation binding experiment using [3H] BK on crude membranes. Dataare from a typical experiment representative of six independent experi-ments with each point measured in triplicate.

dishes and treated with sodium butyrate and/or tetracy-cline for 1, 2 or 3 days. As indicated in Fig. 3, the expres-sion level of the receptor was very low (just above thebackground) in absence of inducer. Tetracycline or sodiumbutyrate alone induced receptor expression but the highestexpression level was obtained with both tetracycline andsodium butyrate (Fig. 3). In contrast to results reportedfor inducible expression of rhodopsin in HEK293S cells[16], optimal-inducible expression was obtained for a 48 hinduction with 2 lg/ml tetracycline and 5 mM sodiumbutyrate, resulting in receptor expression of 5.5 · 106 recep-tor sites/cell. We interpreted the decrease in expressionlevel after 48 h as a result of a toxic effect of sodium buty-rate on cells during prolonged treatments. Interestingly,unlike the loss of expression seen for the constitutiveexpression system, the level of receptor expression obtainedby this inducible system remained constant over the cellpassages tested.

Ligand-binding and functional properties of the receptor

expressed in inducible cell line

Ligand-binding affinities were measured on crude mem-branes prepared from cell line expressing the RB2-6His N-C9C receptor after a 48 h induction with tetracycline andsodium butyrate. As indicated in Fig. 4, the Kd values of

Fig. 3. Expression of the RB2-6His N-C9C receptor in the inducibleHEK293S TetO stable cell line under different conditions. HEK293S cellswere grown to near confluence in 48-well plates (5 · 104 cell/plate) incomplete medium as indicated in Materials and methods. Induction wasinitiated by incubating the cells with fresh medium supplemented asindicated for the different periods of time shown. The data are from arepresentative experiment performed on the clone cell line in which thehighest expression level was obtained. This clone was selected for furthercharacterization. Each bar corresponds to the average amount of sites/cellfrom triplicate culture dishes.

Fig. 4. Binding and activation properties of the RB2-6His N-C9C receptor expressed in the tetracycline-inducible HEK293S stable cell line. Saturationbinding experiments were performed on crude membranes as indicated in Materials and methods. Scatchard plot of a representative binding experimentwith the B2 receptor antagonist [125I]HPP-HOE-140 (performed six times) (a) and the B2 receptor agonist [3H]BK (performed three times) (b). (c)Activation property determined by measuring the total inositol phosphates production induced by increasing amount of BK on intact cells as indicated inMaterials and methods. Data are from a typical experiment representative of three independent experiments, each performed in triplicate.

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the agonist [3H]BK (0.27 nM) and of the antagonist[125I]HPP-HOE (0.36 nM) are similar to the values mea-sured for the B2 receptor in other mammalian cell types[22,23]. Only one saturable-binding site was detected withBmax values in the range of 100–125 pmol/mg of protein,corresponding to a 2-fold increase compared to that ofthe receptor expressed constitutively. Although a single[3H]BK high affinity site was detected, it is not exclude thatvery low affinity sites corresponding to receptors uncoupledto G-protein might exist as already described [25,26] but itsdetection would have required a much higher concentra-tion of ligand. Indeed, it is likely that at this expressionlevel, receptors exceed available G-proteins, although toour knowledge, the G-protein content in HEK293S cellshas not been quantified. We also demonstrated that theagonist stimulated receptor can induce the production ofinositol phosphates with a EC50 value of 1.2 nM which isin the range of values usually reported for the WT B2

receptor expressed in mammalian cells [22,27]. Togethertheses results indicate that the receptor is fully functionaland that the recombinant receptor expressed at the plasma

membrane is correctly folded and signals similar to the WTreceptor obtained from native tissues.

Solubilization and purification of the B2 tagged receptor

from the inducible cell line

The receptor was solubilized from crude plasma mem-branes prepared from cells grown as monolayer in a15 cm dish. The solubilization and purification yields wereestimated by following a fraction of the receptor covalentlylabeled with a radioiodinated ligand. The yield was calcu-lated by measuring at each step, the radioactivity corre-sponding to the covalent ligand–receptor complexes asalready described. A small aliquot of crude plasma mem-branes (1 mg for a total of 70 mg protein used in the exper-iment) was photolabeled with a specific radioiodinated B2

receptor antagonist [125I]HPP-HOE-140 at a concentrationof 1 nM as described in [19]. In these conditions nonspecificcovalent reaction was negligible and undetectable. Photo-labeleled membranes were pooled to the unlabeled mem-branes and the receptor was then solubilized with 1%

Table 2Purification of the tagged B2 bradykinin receptor (RB2-6His N-C9C) fromthe inducible HEK293S cell line

Purificationstep

Receptor(nmol)

Protein(mg)

Specific activity(nmol/mg ofprotein)

Yield (%)

Step Total

Membranes 7 70 0.10 100 100Solubilization 4.9 49 0.10 70 70Hydroxyapatite 2.45 17 0.14 50 351D4-Sepharose 1.34 0.12 11 54 19Ni–NTA 0.67 0.055 12 50 9.5

For this experiment, 15 · 15 cm plates were used, corresponding to6 · 108 cells.

P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311 307

DM. As shown in Table 2, the solubilization yield was70%. We observed that when the solubilized proteins weredirectly bound to rho-1D4-Sepharose gel, the immunoaffin-ity purification step was not very efficient. However, whenthe soluble extract was prepurified on hydroxyapatite theefficiency of the immunopurification step was stronglyincreased. Although hydroxyapatite had a weak efficiencyfor receptor purification (1.4-fold purification) (Table 2),this step was particularly effective in concentrating thesolubilizate (7-fold) without increasing the detergent con-centration that was maintained at 0.5%. After immunoaf-finity purification on rho-1D4-Sepharose, a major diffuseband with a molecular mass of 60–70 kDa was detectedon silver stained SDS polyacrylamide electrophoresis gels(Fig. 5a, lane 1). A minor thin band with higher mass(around 90–100 kDa) and four to five thin minor bandswith molecular masses of 36, 38, 45, and 47 kDa were alsodetected. When analyzed by western blotting with rho-1D4antibody (Fig. 5b, lane 1), the same bands were detected.We suspected that the different bands corresponded to sev-eral glycosylated forms of the receptor. Indeed, it has beenreported that the human B2 receptor is highly glycosylatedin HEK293S [26,27]. Our hypothesis was confirmed bydeglycosylation of the receptor with N-glycosidase F whichresulted in homogenization of the electrophoretic proteinspattern with the appearance of a major band of molecularmass of 35 kDa and a minor band of 60–70 kDa whendetected either by silver staining (Fig. 5a, lane 2) and wes-tern blotting with the rho-1D4 antibody (Fig. 5b, lane 2).As shown in Fig. 5a and b, lane 4; the 35 kDa and 60–70 kDa bands were also detected when the deglycosylatedsample was further purified by Ni–NTA chromatography.Thus, this result clearly indicates that the proteins detectedeither by silver staining and western blot corresponded tothe different forms of the intact B2 receptor bearing theHis tag and C9 tag. Although the 35 kDa protein corre-sponds to a molecular mass lower than the human B2

receptor molecular mass of 41 kDa predicted from itsamino acid sequence, it very likely represents the non-N-glycosylated form of the receptor. Indeed, this unusualmigration is probably due to the hydrophobic characterof the receptor in agreement with others works reportinga similar migration for the unglycosylated human B2 recep-

tor [28–30]. The 60–70 kDa band still present after deglyco-sylation might represent a fraction of receptor resistant toproteolyse but it is also possible that it corresponded to adimeric form of the unglycosylated receptor (see Discus-sion). As shown in Fig. 5a, lane 3, the endoglycanase couldbe readily removed from the purified receptor preparationby adsorbing the sample on Ni–NTA beads; the enzymepassed in the flow through and migrated as a sharp bandjust above the thin band corresponding to the nonadsorbedreceptor.

Autoradiography of the blotted membrane revealed theproteins photolabeled with the antagonist ligand (Fig. 5c).A major 60–70 kDa band was detected and correspondedto the band revealed by silver staining and western blotting(Fig. 5c, lane 1). Two others minor bands were alsodetected, one with a 47 kDa molecular mass and one at100–110 kDa (Fig. 5c, lane 1). Taking into account thatthe covalent reaction was performed on intact cells, the60–70 kDa band represents very likely the mature glycosyl-ated form of the receptor expressed at the cell plasma mem-brane. Indeed, we have previously shown that the ligand[125I]HPP-HOE-140 reacted exclusively with the receptorexpressed at the plasma membrane of COS-7 cells andHEK293 cells [19]. The 47 kDa labeled band detected inFig. 5c, lane 1 might correspond to a degradation productof the B2 receptor mature form or represented a less glycos-ylated form of the receptor able to bind the ligand and highmolecular forms might represent aggregated receptors orreceptor dimers. The 35 kDa band detected by westernblotting and silver staining was also detected by autoradi-ography (Fig. 5c, lane 2), thus confirming that this bandcorresponds effectively to the receptor. However, a 45–47 kDa photolabeled band was also detected in the degly-cosylated sample (Fig. 5c, lane 2). This band was notrevealed with the rho-1D4 antibody (Fig. 5b, lane 2), sug-gesting that it might represent a proteolytic fragment of the60–70 kDa receptor form missing the C9 epitope tag. The60–70 kDa protein detected by western blot in the deglyco-sylated sample (Fig. 5b, lane 2) was not detected by auto-radiography suggesting that this protein cannot reactwith the ligand. As discussed above, this band which likelycorresponded to a receptor form, might represent a recep-tor displaying a very low affinity for the ligand or mightrepresent preformed nonglycosylated receptors dimers.

Quantification of silver stained proteins by densitometryscanning indicated that the 35 kDa and 60–70 kDa recep-tor entities represented 69% and 21%, respectively, of thetotal protein amount of the immunopurified preparation(Fig. 5a, lane 2) and 73% and 17%, respectively, for theNi–NTA purified receptor (Fig. 5a, lane 4). This estimationdemonstrated that the receptor is >90% pure after theimmunoaffinity step when quantified on the deglycosylatedpreparation. Furthermore, these results indicate that theNi–NTA purification step did not increase the purity ofthe receptor but only eliminate the glycosidase from theimmunoaffinity-purified preparation (Fig. 5a, lane 4). Thespecific activity of the purified receptor was 11 nmol/mg

Fig. 5. SDS–PAGE analysis of the purified B2 receptor from the inducible HEK293S cell line. The receptor expression was induced by treatment of thecells grown on 15 cm plates with tetracycline and butyrate as indicated in Materials and methods. The receptor was purified after DM solubilization fromcrude cell membranes as described in Materials and methods. Lane 1: rho-1D4-Sepharose eluate (8 lg), lane 2: 1D4-Sepharose eluate treated with N-glycosidase F (6 lg), lane 3 and 4: Ni–NTA purification of the N-glycosidase F treated rho-1D4-Sepharose eluate, passthrough (5.5 lg) (lane 3), eluate ofNi–NTA beads (4 lg) (lane 4). (a) Proteins stained by silver. In lane 3, two bands migrating in the same molecular weight range are visible, the upper bandis the N-glycosydase F (b) Western blotting with rho-1D4 antibodies as indicated in Materials and methods. Arrows show the positions of the two forms ofB2 receptor. The lower form corresponds to the deglycosylated receptor. (c) Autoradiography of the blotted PVDF membrane shown in (b). Arrow showsthe deglycosylated receptor photolabeled with [125I]HPP-HOE-140, the band corresponds to the lower receptor form detected in (b).

308 P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311

P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311 309

protein when calculated from the radioactivity covalentlyassociated to the receptor and assuming one binding siteper receptor. This value is less than the theoretical specificvalue of 24.5 nmol/mg protein calculated from protein con-tent and the molecular mass of the receptor of 41 kDa. Thisapparent discrepancy likely resulted from the method ofreceptor quantification used. Indeed, when the receptorpatterns obtained by silver stained gel (Fig. 5a, lane 1)and western blot ((Fig. 5b, lane 1) were compared to thepattern obtained by autoradiography (Fig. 5c, lane 1), itwas obvious that the glycosylated receptor forms cova-lently labeled with the radioiodinated ligand, representedless than 50% of the total amount of the purified receptorunder different forms.

Discussion

In this study, the overexpression and purification of thehuman bradykinin B2 receptor is described. We decided tooverexpress in HEK293S cells for several reasons: (1) mam-malian cells offer the advantages of posttranslational mod-ification and proper folding of GPCRs [9,11,31], (2)HEK293S cells can be grown adherently, but also canbe easily adapted to growth in suspension culture thusallowing large scale production in a bioreactor [14], (3)HEK293S cells have been demonstrated to be a good cellsystem for overproduction of others mammalian GPCRssuch as rhodopsin [14,16] and b2 adrenergic receptor [32],which belong to the same GPCR family as that of the bra-dykinin receptors (family A) [3].

We first generated a stable cell line expressing the B2

receptor constitutively. The maximal expression levelobtained with this system was 3 · 106 sites/cell when testedon intact cells, and 60 pmol/mg protein when measured oncrude cell membranes. The recombinant tagged receptorwas well expressed at the plasma membrane and possessedpharmacological-binding and activation properties thatwere expected for a mature and fully functional B2 recep-tor. These pharmacological properties were similar to thoseof the B2 receptor in native tissues [24] or when expressedas a recombinant protein in different mammalian types ofcells [22,23,33]. Although this expression level is one ofthe highest expression level obtained for this receptor, weobserved that the expression level was not stable over timeand decreased with cell passage number. The expressioninstability encouraged us to attempt to overcome this prob-lem by creating a stable cell line in which the expression ofreceptor could be induced. We showed that in the tetracy-cline-inducible cell line, the expression of the tagged B2

receptor was almost undetectable in absence of any indu-cer. The receptor expression was very efficiently inducedby combined addition of tetracycline and butyrate andattained a level of 100 pmol/mg membrane protein. Thesynergistic effect of sodium butyrate and tetracycline wasalready described for the expression of rhodopsin inHEK293S using the same vector [16] and for the b2 adren-ergic receptor using a pGEM-T vector [32]. One can postu-

late that the effect of sodium butyrate likely resulted frominduction of gene expression due to its inhibitory effecton histone deacetylases involved in chromatin remodelingduring gene transcription as already reported [34,35]. Ourresults indicated that the TetR repressor protein is extre-mely effective in blocking receptor expression and that thisinhibition can be strongly reversed by addition of tetracy-cline. Furthermore, the amount of the induced receptorwas reproducible and we observed no decrease of receptorexpression over the cell passages. In that sense, the induc-ible expression strategy presents a guarantee to start solu-bilization and purification experiments with a calibratedexpression receptor level. Moreover, the inducible systemoffers the opportunity to overexpress toxic or unstablereceptors such as constitutively activated receptors asreported for rhodopsin [36]. Our finding that the epitope-tagged B2 receptor (6His at the N-terminus and C9 at theC-terminus) possessed unchanged-binding and activationcharacteristics when tested on intact cells further confirmthat this receptor is well expressed in a mature form atthe plasma membrane.

Successful expression of mammalian membrane proteinsdepends of many factors such as posttranslational modifi-cations, correct folding and intracellular trafficking[10,11,31]. Deficiency or limitation in any of these processesmay lead to production of inactive and/or insoluble proteinwhich may accumulates in intracellular compartments.Western blotting, photolabeling and enzymatic deglycosy-lation experiments demonstrated that the B2 receptorexpressed at the plasma membrane is highly glycosylated.Indeed, the human bradykinin receptor possesses threepotential N-glycosylation sites (N3 and N12 located inthe N-terminus and N180 on the second extracellular loop)[37]. It has been reported that the three sites are glycosyl-ated in COS-7 and HEK293 cells and that glycosylationis required for maximal cell surface expression of the recep-tor [28]. We observed that in HEK293S cells, the absence ofthe N3 site (which has been deleted in our B2 receptor con-structs) is not absolutely required for normal expressionand functionality of the receptor as compared to the WTreceptor. We cannot exclude that the addition of His tagat the N-terminus might favor plasma membrane expres-sion of B2 receptor and compensate the deleterious effectof the loss of the N3 glycosylation site. If so, this effect doesnot depend on the nature of the His-tag itself because iden-tical expression levels were obtained with a c-Myc tagreceptor construct lacking the N3 site. Heterologous over-expression of the B2 receptor has been reported in othereukaryotic cell systems such as baculovirus/Sf9 system withmaximal expression level of 10 pmol/mg protein [30,38],and BHK cell using the Semliki Forest virus vectors withexpression level of 10 pmol/mg protein [23] and 55 pmol/mg protein when 2% of dimethylsulfoxide were added inthe culture medium [39]. Thus, the expression level thatwe obtained in our HEK293S cell line, 100 ± 20 pmol/mgprotein corresponding to 27 lg protein/15 cm plate, is thehighest reported so far for the human bradykinin B2 recep-

310 P. Camponova et al. / Protein Expression and Purification 55 (2007) 300–311

tor and one of highest among GPCRs expressed in eukary-otic cells [11]. We have not been able to increase this levelby addition of dimethylsulfoxide (data not shown) as it hasbeen reported for the B2 receptor expressed in BHK cells[39]. One can postulate that the absence of effect of DMSOin our cell line resulted from the saturation of the synthesisand/or the protein folding and trafficking machineries ofthe cell. Indeed, in BHK cells, the B2 receptor expressionlevel can be increased by 6-fold but was only 10 pmol/mgprotein in absence of DMSO, whereas it was initially10-fold higher in our system. We observed that DM wasefficient for solubilization of the B2 receptor from crude cellmembrane extract. Although DM is considered as a nonde-naturing detergent for membrane proteins [11], weobserved that the functionality of the bradykinin B2 recep-tor was not preserved during the solubilization step andwas not recovered during the purification. However, wedemonstrate that the B2 receptor can be purified to almosthomogeneity by a two-step purification using serialhydroxyapatite and rho-1D4 immunoaffinity columns.We observed that the purified preparation contained sev-eral glycosylated forms of the receptor but could be madehomogeneous by treatment with N-glycosidase F. Indeed,enzymatic deglycosylation resulted in appearance of amajor faint band at 35 ± 1 kDa which likely representsthe nonglycosylated receptor in agreement with othersworks [28–30]. A minor band at 65 ± 4 kDa was alsodetected which may represent the fully glycosylated recep-tor resistant to enzymatic action or may correspond todimeric forms of deglycosylated receptor. Indeed, it hasbecome clear over the last ten years that GPCRs formhomo or hetero oligomers and it has been reported thatthe B2 receptor formed homodimers when expressed inPC-12, CHO-K1 and HEK293 cells [29,40]. The combina-tion of western blotting, silver staining analysis and proteindetermination altogether demonstrate that the receptor ishighly pure (>90%) after the rho-1D4 immunopurificationstep. This work demonstrates that we can produce 8 lg ofpure receptor/15 cm plate. Preliminary assays on suspen-sion culture in 500 ml flask indicate that we can attaineda cellular density of 2 · 106 cell/ml and produce 160 lg ofpure receptor from 1 liter of culture medium. This level isone of the highest level obtained for a GPCR in a eukary-otic system and the highest expression level for the B2 bra-dykinin receptor [11,41]. Moreover, to our knowledge, thisis the first report of the purification of the B2 bradykininreceptor to near homogeneity from a cell system over-expressing this receptor. The quantities of pure receptorthat we routinely obtained, will allow us to test conditionsfor its renaturation by reconstitution in liposomes or deter-gent lipids mixed micelles. Finally, the scale up of suspen-sion culture of our stable cell line in a bioreactor is underprogress. This should dramatically improve the receptorexpression level as it has been observed for rhodopsin (Phi-lip J. Reeves, personal communication) and will likelyallow production of the amount of biomass for purificationof the mg amounts of protein required for structural stud-

ies that are planned. In conclusion, we have successfullydeveloped a procedure for overexpression of the humanbradykinin B2 receptor and designed a protocol for its effi-cient purification. This process should enable us to pro-gress rapidly towards the reconstitution of a functionalreceptor required for structural characterization of thisreceptor.

Acknowledgments

This work was supported by Institut National de laSante et de la Recherche Medicale (INSERM), the CentreNational de la Recherche Scientifique (CNRS), the Minist-ere de la Recherche (ACI ‘‘Molecules et Cibles Therapeu-tiques’’ n�355) and the Fondation pour la RechercheMedicale. We are grateful to Jean-Francois Guichou andAlain Chavanieu for the synthesis of the C9 peptide. Wethank Dr. Philip J. Reeves (University of Essex, Colchester,UK) for the generous gift of HEK293S TetR cells.

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