Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris

Protein Expression and Purification 55 (2007) 1–8 www.elsevier.com/locate/yprep

Heterologous expression and characterization of the recombinant bradykinin B2 receptor using the methylotrophic yeast Pichia pastoris Arun Kumar Shukla a

a,*

, Winfried Haase b, Christoph Reinhart a, Hartmut Michel

a,*

Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany b Department of Structural Biology, Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438 Frankfurt am Main, Germany Received 6 December 2006, and in revised form 14 February 2007 Available online 24 March 2007

Abstract The human bradykinin subtype 2 receptor (B2R), a member of class A GPCRs, was heterologously expressed in the methylotrophic yeast Pichia pastoris. The recombinant receptor was produced as a fusion protein with affinity tags and it was expressed at a level of 3.5 pmol/mg of total membrane protein. [3H]Bradykinin binding analysis revealed that the recombinant receptor binds to its endogenous ligand bradykinin with high affinity (Kd = 0.87 ± 0.1 nM), similar to the native receptor. Enzymatic deglycosylation revealed that the recombinant B2R was produced in a glycosylated form. Immunogold staining of the Pichia cells expressing B2R suggested that the recombinant receptor was localized intracellularly and it was not present in the plasma membrane. The data presented here should facilitate isolation of the recombinant receptor for structural studies.  2007 Elsevier Inc. All rights reserved. Keywords: GPCR; Bradykinin receptor; Heterologous expression; Glycosylation; Localization

G-protein-coupled receptors (GPCR)1 constitute the largest family of the cell surface receptors and they are activated by a variety of ligands such as light, hormones, peptides, neurotransmitters, nucleotides and odorants [4]. GPCRs are involved in many cellular and physiological processes [19] and represent an important class of pharmaceutical drug targets [10]. However, due to lack of structural information, structure based drug design on GPCRs has not been possible. Structural studies on GPCRs, like other membrane proteins, remain challenging, particularly due to their low expression level in the native tissues. To

*

Corresponding authors. Fax: +496963031002. E-mail addresses: [email protected] (A.K. Shukla), [email protected] (H. Michel). 1 Abbreviations used: B2R, bradykinin subtype 2 receptor; GPCR, Gprotein-coupled receptor; EndoH, endoglycosidase H; PNGaseF, peptideN-glycosidase F; P. pastoris, Pichia pastoris. 1046-5928/$ - see front matter  2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pep.2007.02.021

overcome this problem, a number of heterologous expression systems have been used to produce large amounts of recombinant GPCRs [25]. In recent years, the methylotrophic yeast Pichia pastoris has become an increasingly popular system for the overexpression of recombinant proteins [13]. Overexpression of several GPCRs has been achieved using this system [25,13,2]. The advantages of P. pastoris system include easy genetic manipulations, growth of the cells up to high cell density in inexpensive media and its ability to perform post-translational modifications. Based on the previous reports of successful production of recombinant GPCRs in P. pastoris, we attempted to express the human B2R as a recombinant protein in this system. The human B2R belongs to the rhodopsin family of GPCRs and it has been extensively studied for its functional relevance [12]. B2R mediates most of the cellular and physiological actions of bradykinin (e.g. contraction and relaxation of smooth muscles, effects on epithelial ion transport, circulation and blood pressure) [21,8]. There

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are several reports suggesting pathophysiological roles of bradykinin, and it makes the selective B2R modulators potential drugs [18]. Structural information on B2R should lead to a better understanding of its ligand binding and activation mechanism. However, direct structural characterization of this receptor remains elusive so far, mainly because B2R is present in small amounts (inadequate for structural analysis) in natural tissues [33,27]. Expression of recombinant B2R in mammalian cells and insect cells have been reported previously, mainly for the purpose of functional characterization [16,24]. We have also attempted heterologous overexpression of the recombinant B2R in mammalian cells and insect cells for large-scale production [32a–c]. However, as the expression levels are only low or moderate, isolation of milligram quantities of B2R using mammalian cells and insect cells remains time consuming and expensive. Therefore, we decided to investigate if heterologous overexpression of B2R in yeast-based system can provide sufficient amounts of this receptor for structural characterization. In this study, we used the methylotrophic yeast P. pastoris for heterologous expression of an affinity tagged recombinant B2R. The recombinant receptor was expressed at significantly higher levels compared to the native tissues and it was characterized with regard to its ligand binding properties, glycosylation and localization. Materials and methods Materials Pichia pastoris strain SMD 1163 was obtained from Invitrogen (San Diego, California, USA), components of Yeast growth media were purchased from Difco (MD, USA) and Sigma (Taufkirchen, Germany). G418 was from Calbiochem (Dermstadt, Germany), DMSO was from Roth (Karlsruhe, Germany) and Glass beads (0.5 mm) were from Euler Prozesstechnik (Frankfurt, Germany). [3H]Bradykinin (60–90.0 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA, USA). Restriction enzymes, PNGaseF and EndoH were from MBI fermentas (St. Leon-Rot, Germany). Immobilon-P polyvinylidene difluoride (PVDF) membranes were from Millipore (Bedford, MA, USA). Anti-his antibody, alkaline phosphatase-coupled streptavidin and alkaline phosphatase-coupled secondary antibody were from Sigma (St. Louis, MO, USA). Construction of recombinant expression vector A PCR was performed with primers B2R_Fw (5 0 -CG GGATCCCTCAATGTCACCTTGCAAGGGCCC-3 0 ) and B2R_Rv (5 0 -CGGAATTCCTGTCTGCTCCCTGCC CAGTCC-3 0 ), using pcDNA3-B2R as a template. The resulting DNA fragment was digested with BamHI and EcoRI restriction enzymes and ligated into appropriately digested pPIC9K (modified) expression vector. The modi-

fied pPIC9K vector has been described earlier [35,7,2]. This vector contains a Flag tag and a His10 tag at the N-terminus and a biotinylation domain of Propionobacterium shermanii transcarboxylase [5] at the C-terminus of multiple cloning site for immunodetection and purification. The tags are separated from B2R coding region by TEV (tobacco etch virus) protease cleavage site. The construct was verified by DNA sequencing (SeqLab GmbH, Germany). Pichia pastoris culture and expression SMD 1163 (his4, pep4, pbr), a protease-deficient strain of P. pastoris was transformed with the recombinant B2R expression construct (1500 V, 25 lF and 600 X). His+ recombinant clones were selected on MD plates (1.34% yeast nitrogen base without amino acids, 0.00004% biotin, 2% dextrose, 1.5% agar) and subsequently, multicopy transformants were selected on YPD-G418 plates (1% yeast extract, 2% peptone, 2% dextrose, 2% agar and 0.025–0.5 mg/ml G418). For expression analysis, six different clones growing at different G418 concentrations were checked by Western blot and the best expressing clone was selected for further experiments. Selected clones were grown (12–16 h, OD600 2–6) in BMGY medium (1% yeast extract, 2% peptone, 1.34% yeast nitrogen base without amino acids, 0.00004% biotin, 1% v/v glycerol, 0.1 M phosphate buffer, pH 6.0). For expression of the recombinant receptor, cells were harvested (2000g, 5 min, room temperature) and resuspended in BMMY medium (same as BMGY, but 0.5% methanol instead of glycerol) to an OD600 of 1. Small-scale cultures were grown in Erlenmeyer flask (20 ml culture in 100 ml flask) at 22 C or 30 C with shaking at 220 rpm. For optimization of the recombinant receptor expression, different concentrations (v/v) of DMSO were added to the P. pastoris culture during methanol induction step. Membrane preparation For preparation of membranes, cells were harvested (3000g, 5 min, 4 C), washed in cold breaking buffer (50 mM sodium-phosphate buffer, pH 7.4, 100 mM NaCl, 2 mM EDTA and complete protease inhibitor cocktail) and resuspended in breaking buffer. Equal amount of glass beads was added to the suspension and cells were broken by vigorous vortexing at 4 C for 5–10 min. The crude lysate was centrifuged (3000g, 10 min, 4 C) in order to remove the nuclei and cell debris. Subsequently, the membranes were pelleted by ultracentrifugation (100,000g, 1 h, 4 C). The membrane pellet was resuspended in storage buffer (breaking buffer supplemented with 5% glycerol) at a protein concentration of 5–10 mg/ml, flash-frozen in liquid nitrogen and stored at 70 C until used. The protein concentration was determined using the bicinchoninic acid reagent (Pierce, Rockford, IL, USA) with BSA as standard.

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[3H]Bradykinin binding assay [3H]Bradykinin binding assay was performed using a standard protocol. Briefly, the membranes were diluted in binding buffer (50 mM Hepes, pH 7.4, 100 mM NaCl, 5 mM EDTA and complete protease inhibitor cocktail). For single point measurements, membranes (5–20 lg of total protein per assay point) were incubated with 10 nM [3H]bradykinin for 45–60 min at 4 C. For saturation binding analysis, different concentrations of [3H]bradykinin (0.1–25 nM) were used. Non-specific interactions were determined in the presence of 1 lM bradykinin. After incubation, the reaction was terminated by rapid filtration through GF/C glass-fiber filters which were presoaked in 0.3% (v/v) polyethyleneimine. Filters were quickly washed four times with ice cold binding buffer and subsequently transferred to counting vials. Radioactivity was measured by liquid-scintillation counting. Dissociation constant (Kd) and maximum expression level (Bmax) was calculated with the ‘‘KaleidaGraph’’ software by nonlinear regression using a single site model. Immunoblot analysis and deglycosylation For immunoblot analysis, proteins were separated by 10% SDS–PAGE and subsequently transferred to a PVDF membrane as described previously [34]. PVDF membranes were blocked with 5% (w/v) nonfat dry milk powder in TBST buffer (25 mM Tris, 192 mM glycine, 20% methanol, 0.1% Tween 20) for 1 h at room temperature. The anti-Flag M2 antibody, the alkaline phosphate-conjugated secondary antibody and the alkaline phosphatase-coupled streptavidin were used according to the manufacturer’s protocol (Sigma). Blots were developed in 10 ml of alkaline phosphatase buffer (100 mM Tris/HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl2) containing 66 ll of BCIP (5-bromo-4chloro-3-indolyl phosphate-p-toluidinium salt, stock solution 50 mg/ml in dimethylformamide) and 33 ll of NBT (nitro blue tetrazolium chloride, stock solution 50 mg/ml in 70% dimethylformamide). Enzymatic deglycosylation was performed by incubating 50 lg of membranes (5 mg/ml) with 1–2 U of PNGaseF or EndoH at 37 C for 2 h. Subsequently, immunoblot analysis was performed, as described above. Immunogold staining Pichia pastoris cells expressing the B2R fusion protein were fixed with 4% paraformaldehyde (in 0.1 M sodium cacodylate buffer, pH 7.2, containing 0.8 M sorbitol, 1 mM MgCl2, and 1 mM CaCl2) for 3 h, room temperature. Then, the cells were washed and treated with 1% sodium metaperjodate. Subsequently, the cells were washed with water and then incubated overnight in 0.05 M NH4Cl. Afterwards, the cells were dehydrated with ethanol, infiltrated with LR White resin (London Resin Company Ltd., Reading, England) and polymerized in gelatine cap-

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sules at 56 C for about 30 h. Thin sections were cut with an ultramicrotome (Ultracut, Reichert, Austria), placed on Formvar coated Nickel grids and used for immunogold labeling. Immunogold labeling was performed on the sections using the following solutions (in same order)—saturated sodium metaperjodate, water, 2% glycine in PBS, PBS, PBS + 1% BSA + 0.1% Tween 20, PBS + 0.1% BSA + 0.05% Tween 20, primary antibody (anti-Flag M2) diluted in PBS + 0.1% BSA, PBS, secondary antibody [goat anti-mouse coupled to 10 nm gold particles (Amersham)]. Subsequently, the sections were washed, treated with 1% glutardialdehyde/PBS, washed with water and then double contrasted using uranyl acetate and lead citrate. Sections were analyzed using an EM208S electron microscope (FEI Company) equipped with a TVIPS 1K · 1K slow-scan CCD camera (Tietz, Munich, Germany). Results Recombinant construct for expression of the B2R For the production of human B2R in P. pastoris, pPICK9K based recombinant expression construct was used (described in [2]). This construct is schematically depicted in Fig. 1. Here, production of the recombinant receptor is driven by a strong alcohol oxidase promoter (AOX1). The activity of this promoter is dependent on the carbon source in the culture medium and maximum activity can be achieved using methanol as a sole carbon source. For membrane targeting of the recombinant B2R, an a-factor signal sequence of Saccharomyces cerevisiae was fused at the N-terminus of the receptor. A Flag tag and a His10 tag at the N-terminus and the biotinylation domain of P. shermanii transcarboxylase [5] at the C-terminus of B2R were also engineered. These tags can be used for immunodetection and purification of the recombinant receptor. In addition, a positive effect of the biotinylation domain on the expression of dopamine D2S receptor in P. pastoris has been reported earlier [7]. The receptor-coding region is separated from the tags by two TEV protease cleavage sites, which provide a possibility for the removal of the tags, if required. A protease deficient strain SMD 1163 was used for expression in order to avoid the proteolytic cleavage of the recombinant receptor. Our previous experience with other GPCRs has suggested that the protease deficient strain SMD 1163 is the most suitable choice. We analyzed six different clones, which were grown on increasing concentration of G418 (0.025–0.25 mg/ml) by Western blot analysis. It appeared that the highest expression level as revealed by Western blot was not obtained from a clone growing on the highest antibiotic concentration and thus not containing the greatest expression cassette copy number. Similar effects were observed for the 5HT5 receptor produced in P. pastoris [35]. The clone growing at 0.075 mg/ml concentration of G418 yielded the best signal on the Western blot and this clone was used

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AOX1

α-factor

Flag His10 TEV

B2R

TEV

Bio

Fig. 1. Schematic representation of the recombinant construct for heterologous production of the human B2R in P. pastoris. AOX1, alcohol oxidase gene 1 promoter; a-factor, coding region for the prepropeptide of the Saccharomyces cerevisiae mating type factor; Flag, coding region for the Flag epitope; His10, Histidine 10 tag; B2R, coding region for the human B2R; TEV, tobacco etch virus protease cleavage site; Bio, biotinylation domain of Propionobacterium Shermanii transcarboxylase.

for further characterization. A time-course analysis of recombinant receptor expression was performed, which revealed maximum expression at 24 h post-induction (data not shown). Therefore, for further experiments, the cells were harvested 24 h post-induction and subsequently membranes were prepared. Immunoblot analysis of the recombinant B2R The expression and the integrity of the recombinant receptor were analyzed by Western blot using either antiFlag antibody (detects the N-terminal Flag tag) or alkaline phosphatase-conjugated streptavidin (detects the C-terminal bio tag). As depicted in Fig. 2 (A and B, lane 2, black arrow head), both anti-Flag antibody and alkaline phosphatase-conjugated streptavidin recognized a major band of 55 kDa. The size of this band corresponds well to the calculated molecular mass of the B2R fusion protein (51.3 kDa). Staining of the recombinant receptor band by alkaline phosphatase-conjugated streptavidin suggested that in vivo biotinylation of the bio domain took place. In addition to the main band at 55 kDa, a band at the size of 65–70 kDa (indicated by grey arrow head) was also detected, which might represent the unprocessed form of the receptor (i.e. processing of the a-factor sequence did not occur).

receptor. As shown in Fig. 3, the ligand binding was saturable and a single binding site was observed. An expression level of 0.5 ± 0.1 pmol recombinant receptor per mg of total membrane protein was observed based on the radio ligand binding experiment. The recombinant B2R binds to [3H]bradykinin with high affinity (Kd = 0.87 ± 0.1 nM) and the Kd value observed here for the recombinant receptor is in good agreement with that of B2R in native tissues [27,28]. Non-specific binding was determined in presence of 1 lM bradykinin and it was less than 10% of total binding. Expression optimization of the recombinant B2R As mentioned above, [3H]bradykinin binding revealed an expression level of 0.5 pmol of recombinant B2R per mg of total membrane protein. A recent study reports that the functional expression level of GPCRs in P. pastoris can be significantly improved by adding DMSO in the culture medium [2]. Therefore, different concentrations of DMSO were added in the Pichia cultures during the methanol induction step. Addition of DMSO resulted in significant increase in the functional expression level of recombinant B2R (Fig. 4). The maximum expression level was observed

Saturation binding analysis of the recombinant B2R A saturation binding experiment using [3H]bradykinin was performed in order to calculated the Bmax (maximum expression level) and the Kd (affinity) of the recombinant M(kDa)

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2

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175 83 62 48

33 Fig. 2. Immunoblot analysis of membranes from P. pastoris cells expressing the recombinant B2R. Immunoblot was developed either using anti-Flag M2 antibody (A) or alkaline phosphatase-coupled streptavidin (B). Lane 1, membranes from control P. pastoris cells (transformed with empty pPIC9K vector) and lane 2, 10 lg membranes from P. pastoris cells expressing the recombinant B2R.

Fig. 3. Saturation binding analysis using [3H]bradykinin on membranes prepared from the P. pastoris cells expressing recombinant B2R. Each point is an average of two independent experiments, each performed in triplicates. The graph was generated by nonlinear regression in KaleidaGraph software using a single site model.

Foldlig and binding

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En d

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tre ate d as eF

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5 4 3 2 1 0

0

1

2

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DMSO (%) Fig. 4. Effect of DMSO on the functional expression of recombinant B2R. Different concentrations of DMSO were added to the P. pastoris culture during methanol induction step. Subsequently, membranes were prepared and [3H]bradykinin binding assay was performed. Each point represents an average of three independent experiments.

in the presence of 2% DMSO and it corresponds to 3.5 pmol/mg (i.e. 0.1 mg of recombinant receptor per litre culture). This expression level is notably higher compared to the expression level of B2R in native tissues [27,33] or that obtained by transient transfection in mammalian cells [16].

Fig. 5. Glycosylation analysis of the recombinant B2R expressed in P. pastoris. Enzymatic deglycosylation was performed on membranes (20 lg) using either PNGaseF (1.0 U) or EndoH (1.0 U). Immunodetection was performed using anti-Flag M2 antibody.

in the perinuclear membranes (Figs. 6C and D). There was no significant staining observed in the plasma membrane although the receptor is a natural resident of the plasma membrane. It is important to mention that similar localization of B2R was observed in both DMSO treated and non-treated cells indicating this effect is not due to addition of DMSO in the culture medium. Discussion

Glycosylation analysis B2R contains three putative N-linked glycosylation sites (i.e. N3, N12 and N180). In order to determine the glycosylation state of the recombinant receptor, enzymatic deglycosylation using PNGaseF and EndoH was performed. PNGaseF cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins while EndoH cleaves within the chitobiose core of high mannose and some hybrid oligosaccharides from N-linked glycoproteins [14]. As shown in Fig. 5, the size of the 55 kDa band was reduced in response to enzymatic deglycosylation (indicate by black arrow head). This result suggested that the 55 kDa band represents glycosylated form of the recombinant receptor. Additionally, the size of the unprocessed receptor was also reduced (indicate by grey arrow head). Receptor localization To investigate the cellular localization of the recombinant receptor, post-embedding immunogold staining experiment was performed on P. pastoris cells expressing B2R. Similar to previous studies [29,27], stacked membranes were observed in P. pastoris cells expressing the recombinant receptor (compare Figs. 6A and B). We also observed that the recombinant receptor was localized mainly intracellularly, either in the membrane stacks or

A number of GPCRs have been expressed in different yeast systems such as S. cerevisiae, Schizosaccharomyces pombe and P. pastoris [22]. Out of these, P. pastoris is of special interest as it can be used for high density fermentation in order to produce large amounts of a given GPCR. Large-scale production of the human ETB endothelin receptor in P. pastoris by high density fermentation has been reported earlier [30]. Other GPCRs which have been successfully expressed in P. pastoris include the human b2-adrenergic receptor and the mouse 5HT5A receptor [35], the human dopamine D2S receptor [7], the human CB1 cannabinoid receptor [9] and the human l-opioid receptor [26]. Here, we tested this system for the heterologous expression of human bradykinin B2 receptor. The recombinant B2R was produced as a fusion protein with affinity tags (Flag tag and His10 tag at the N-terminus and Bio tag at the C-terminus) to facilitate immunodetection. A positive effect of S. cerevisiae a-factor on the expression of 5HT5A receptor in P. pastoris has been reported previously [35] and therefore, this sequence was fused at the N-terminus of B2R coding region. The biotinylation domain at the C-terminus of B2R was used because a previous study indicates that fusion of the biotinylation domain exerts a positive effect on the expression of human dopamine D2S receptor in P. pastoris [7]. Immunoblot analysis of the membrane from P. pastoris cells expressing B2R revealed a major band of 55 kDa. The size of this band corresponded closely to the calculated

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Fig. 6. Immunogold staining of P. pastoris cells expressing the recombinant B2R. Post-embedding labeling was performed using anti-Flag M2 antibody and gold-coupled goat anti-mouse secondary antibody. Gold particles were present mainly in the intracellular membranes (B, C and D). P. pastoris cells transformed with empty pPIC9K vector were used as a negative control (A). N, nucleus; M, mitochondria; P, peroxisome. Scale bars represent 0.5 lm.

size of B2R fusion protein. Staining of this band by both anti-Flag and streptavidin indicated that it represents the full-length receptor (Flag and Bio tag are present at the N- and C-terminus, respectively). An additional band of 65–70 kDa was also observed that corresponds to the unprocessed receptor population. In principle, a-factor should be removed from the B2R fusion protein by the signal peptidase (in the endoplasmic reticulum) and Kex2 protease (in the Golgi apparatus). Partial processing of the recombinant receptor has been previously reported in the case of b2-adernergic receptor and 5HT5A receptor [35], the human ETB endothelin receptor [29] and the human dopamine D2S receptor [7]. The possible reasons for this partial processing of the receptor include glycosylation of the B2R at the N-terminus (it might hinder with Kex2 protease action) or retention of a fraction of the recombinant receptor in endoplasmic reticulum.

Saturation binding analysis using [3H]bradykinin revealed an expression level of 0.5 pmol/mg for the recombinant B2R which was further increased to 3.5 pmol/mg by addition of DMSO in the culture medium. This expression level of B2R is significantly higher than previously reported levels of B2R in native tissues (100–300 fmol/mg). More importantly, the recombinant receptor binds to its endogenous ligand bradykinin with high affinity and the Kd value of the recombinant B2R is similar to that of B2R in native tissues [27,6,23]. This result suggests that the tags introduced at the N- and C-terminus of B2R had no deleterious effect on its ligand binding properties. Use of DMSO to improve the functional expression of recombinant GPCRs in the methylotrophic yeast P. pastoris has been published recently [2]. Out of the 20 selected GPCRs, DMSO could improve the functional expression of 16 GPCRs up to sixfold. The exact reason of this DMSO effect is not yet clearly

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understood. DNA microarray based analysis shows that addition of DMSO results in increased phospholipid biosynthesis and cellular membrane proliferation in S. cerevisiae [17]. DMSO is also known to alter the expression pattern of yeast [38] and it increases membrane permeability in some cases [37]. Further studies are required to get a detail insight into the DMSO induced increase in expression level of recombinant GPCRs. B2R exists as a protein of 69 kDa in human fibroblastic cells [20,1,3] and it might represent hyperglycosylated form of the receptor (the B2R contains three putative Nlinked glycosylation sites). It has been reported that all three sites are glycosylated, most probably through complex type oligosaccharides side chains containing terminal sialic acid residues [36]. Glycosylation of B2R does not affect its ligand binding properties but it is important for proper targeting of the receptor [15]. During this study, enzymatic deglycosylation (using PNGaseF and EndoH) of the recombinant B2R resulted in increased mobility of the receptor. It suggests that the recombinant B2R produced in P. pastoris was glycosylated. However, hyperglycosylation like that in human fibroblast cells was not observed. Appearance of partially glycosylated receptor might represent the saturation of rate limiting step in the carbohydrate processing of P. pastoris cells due to overexpression of the recombinant receptor. In order to investigate the localization of the recombinant receptor, we performed post-embedding immunogold labeling experiment on P. pastoris cells expressing the recombinant B2R. Expression of recombinant B2R resulted in the appearance of stacked membranes. Similar stacks of membranes have been observed in P. pastoris cells expressing the human ETB endothelin receptor [29] and the human dopamine D2S receptor [7]. These membrane stacks resemble the so called Karmellae of S. cerevisiae [11,31]. The origin and assembly of these membrane stacks is not well understood and need further investigation. Interestingly, the recombinant receptor was mainly confined into these stacked membranes and perinuclear membranes. Similar intracellular retention of the human b2-adrenergic receptor [34], the human ETB endothelin receptor [29] and the human dopamine D2S receptor [7] in Pichia cells has been observed. High-level expression of recombinant GPCRs often leads to intracellular accumulation of the recombinant receptors probably due to the saturation of trafficking machinery of the host cell. However, the intracellularly retained recombinant B2R binds to [3H]bradykinin with high affinity. In conclusion, we have established heterologous expression of an affinity tagged human B2R in the methylotrophic yeast P. pastoris. The recombinant receptor exhibited high affinity binding to its cognate ligand bradykinin, similar to the B2R in native tissues. The recombinant B2R was glycosylated and localized mainly in the intracellular membranes. This yeast-based expression of B2R should facilitate further characterization of the receptor.

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