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Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination
Methods 55 (2011) 281–286
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Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination Mitsunori Shiroishi a,b, Takuya Kobayashi a,c, Satoshi Ogasawara c, Hirokazu Tsujimoto a,c, Chiyo Ikeda-Suno a,c, So Iwata a,c,d,e, Tatsuro Shimamura a,c,⇑ a
Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan d Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK e Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK b c
a r t i c l e
i n f o
Article history: Available online 27 August 2011 Keywords: GPCR Expression Pichia pastoris Yeast Structure Membrane protein
a b s t r a c t G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H1 receptor (H1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H1R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction G-protein coupled receptors (GPCRs) are one of the largest family of membrane proteins and more than 800 GPCRs are identified in human [1]. GPCRs play essential roles in the signal transduction pathway by receiving extracellular signals and activating G-proteins to relay the signal inside the cell. Reflecting the pivotal functions, GPCRs constitute 50% of all drug targets. Until 2007, only rhodopsin structures [2–4] were available to the structural modeling of other GPCRs for effective development of new drugs. Rhodopsin is the exceptional GPCR because the receptor is stabilized by covalently bound retinal, and can be purified abundantly from retina for the structure determination. In contrast, most GPCRs are expressed at low level and difficult to purify from the native tissues. Therefore, for the GPCR structural studies, it is essential to establish the overexpression system of stable receptors. However, recombinant production of functional GPCRs is known to be ⇑ Corresponding author at: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan. Fax: +81 75 753 4660. E-mail address: [email protected] (T. Shimamura). 1046-2023/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2011.08.015
difficult partly due to their conformational flexibility [5]. Five non-rhodopsin GPCR structures have been determined so far using the stabilized receptors by protein engineering. They are human b2-adrenergic receptor (b2AR) [6,7], human adenosine A2A receptor (A2AAR) [8], human CXCR4 chemokine receptor [9], human dopamine D3 receptor [10] and turkey b1-adrenergic receptor (b1AR) [11]. The structure of b1AR was determined by the vapor diffusion crystallization method using the thermostabilized multiple mutant. As for b2AR, one structure was determined as a Fab complex by the vapor diffusion crystallization method [6]. The other structure was determined from the crystals formed in lipidic cubic phase [7]. In this case, the flexible third cytoplasmic loop was replaced with T4-lysozyme (T4L) [12]. The latter strategy was successfully applied to the structural studies of other human GPCRs [8–10]. Though the stabilization strategies were different, however, these receptors were all overexpressed in insect cells and the availability of other hosts for the GPCR structural study was unknown. Human histamine H1 receptor (H1R) is involved in allergic responses and the target of the antihistamine drugs [13–15]. Recently, we succeeded to determine the structure of H1R at 3.1 Å resolution using lipidic cubic phase crystallization method [16].
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The receptor construct contained a replacement of the third cytoplasmic loop with T4L (H1R-T4L) and was expressed in Pichia pastoris. Here we describe the method how we established the overexpression system of H1R in yeast and purification system of the receptor used for the structure determination [16].
the T4L insertion site and the deletion length in N and C terminal regions. However, it is sometimes necessary to prepare and screen several constructs as were done in the structural studies of other GPCRs [7,9,10,12]. 3. Expression of H1R-T4L in P. pastoris
2. Design of the H1R-T4L construct Fig. 1a shows the construct of H1R-T4L used for the expression in P. pastoris. The construct consists of H1R with a N-terminal 19residue deletion and a replacement of the third cytoplasmic loop by T4L, tobacco etch virus (TEV) protease cleavage site, green fluorescent protein (GFP) and octa-histidine (His8) tag. This construct was designed based on the structural information of three nonrhodopsin GPCRs (b1AR [11], b2AR [7] and A2AAR [8]) available at that time. In these structures, N- and C-terminal regions were disordered (Fig. 1b and c), suggesting that these regions are flexible and could affect badly for the expression of GPCRs [17]. Therefore, we truncated the N-terminal 19 residues (Met1-Lys19) of H1R. It should be noted that the truncated region contains two N-linked glycosylation sites (Asn5 and Asn18) that can be obstacles to crystal packing [18]. The C-terminal region of H1R was proposed to be very short after the helix VIII by the alignment (Fig. 1c) and we did not delete any residues at C-terminal region. We replaced the third cytoplasmic loop between 5th and 6th transmembrane helices with T4L at the corresponding site to b2AR that is an aminergic receptor similarly with H1R. GFP was fused to the receptor for the evaluation of monodispersity by fluorescence size exclusion chromatography (FSEC) [19]. His8-tag was tagged for the affinity purification at the C-terminal of GFP. GFP and His8-tag were removed at the final step of the purification procedure by TEV protease cleavage. Consequently, H1R-T4L showed better expression level, binding activity to pyrilamine, and FSEC profile than the full-length H1R when expressed in Saccharomyces cerevisiae, suggesting that the stability of the receptor was increased by these modifications (Shiroishi, Unpublished data). The construct was used for the structural determination without further optimizing
In this study, we used two yeast species, S. cerevisiae and P. pastoris. P. pastoris was used for the expression of H1R-T4L because it generates large biomass in fermentor cultures [5]. S. cerevisiae was used for the construction of the H1R-T4L gene because the plasmid was easily and quickly assembled by yeast homologous recombination of PCR fragments [20,21]. 3.1. Construction of the H1R-T4L gene in S. cerevisiae The template DNA encoding full-length H1R was codon-optimized to yeast and synthesized. Three PCR fragments encoding Thr20-Cys221 of H1R, Asn2-Tyr161 of T4L and Leu405-Ser487 of H1R, respectively, were amplified using appropriate primers and templates by the standard PCR using KOD plus DNA polymerase (TOYOBO, Japan). A 30–40 ng of SmaI-linearized plasmid pDDGFP2 [20] and 3 ll of the PCR reaction mixtures were co-transformed into S. cerevisiae strain FGY217 [20,22]. Transformants harboring the plasmid encoding H1R-T4L were selected on–Ura–agar plates [0.192% (w/v) yeast synthetic drop-out media without Ura (Sigma), 0.67% (w/v) yeast nitrogen base without amino acids (BD), 2% (w/v) agar and 2% (w/v) glucose]. The S. cerevisiae transformant was cultured in 5 ml of–Ura medium [0.192% (w/v) yeast synthetic dropout media without Ura, 0.671% (w/v) yeast nitrogen base without amino acids and 2% (w/v) glucose] at 30 °C for 22–24 h. The generated plasmid encoding H1R-T4L was isolated from S. cerevisiae with the Miniprep Kit (Qiagen) by disrupting cells with 0.5 mm glass beads. The plasmid was amplified in Escherichia coli strain DH5a, and the DNA sequence for H1R-T4L was verified by DNA sequencing. Coding regions of H1R-T4L followed by TEV protease recognition site, GFP and His8-tag were amplified by PCR using a
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Fig. 1. Design of H1R-T4L construct. (a) Schematic representation of H1R-T4L construct. Residues 222-404 of H1R were replaced by T4L. The H1R-T4L is connected to His8tagged GFP by the TEV protease recognition sequence. (b) Sequence alignment of N-terminal region between H1R, b2AR and A2AAR. Conserved residue was shown in red. Disordered residues in b2AR and A2AAR structures are shown in grey. The truncated residues of H1R were shown in green. (c) Sequence alignment of C-terminal region. (d) Sequence alignment around T4L insertion region.
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forward primer containing a BamHI site (50 -CTA GAA CTA GTG GAT CCA CCA TG-30 ) and a reverse primer containing an EcoRI site (50 GCT TGA TAT CGA ATT CCT GCA GTT AAT G-30 ). The PCR products were digested with BamHI and EcoRI, and subcloned into the pPIC9K vector. 3.2. Integration of the H1R-T4L gene into the genome of P. pastoris The PmeI linearized pPIC9K vector prepared in Section 3.1 was transformed into the P. pastoris SMD1163 strain by electroporation (2000 V, 25 lF, and 600 X) using a Gene Pulser I (Bio-Rad). Then, we selected the clone with the highest expression level and used for the large-scale expression. Clone selection was performed on the MD-agar plate [1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 2% (w/v) glucose and 1.5% (w/v) agar], followed by YPD-agar plate containing 0.1 mg/ml geneticine. To examine the expression level of H1R-T4L, the clones were cultured at small-scale as follows. A single colony of P. pastoris transformant on the YPD plate with 0.1 mg/ml geneticine was inoculated into 5 ml of BMGY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 1% (w/v) glycerol, 0.1 M phosphate buffer at pH 6.0] at 30 °C with shaking at 250 rpm until an OD600 of 2–6 was reached. The cells were harvested by centrifugation. To induce expression, the cell pellet was resuspended to an OD600 of 1.0 in 5 ml of BMMY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 0.5% (v/v) methanol, 0.1 M phosphate buffer at pH 7.0] containing 2.5% (v/v) DMSO at 30 °C. The addition of DMSO has been known to improve the expression level of GPCRs in P. pastoris [23]. Cells were harvested within 20–22 h after induction. Yeast cells were suspended in 500 ll of the breaking buffer [50 mM HEPES pH 7.5, 120 mM NaCl, 5%(v/v) glycerol, 2 mM EDTA and EDTA-free protein inhibitor cocktail (Roche)]. Cells were disrupted with 500 ll of 0.5 mm glass beads by vortexing at 2500 rpm in 2 ml centrifuge tube. Undisrupted cells and cell debris were separated by centrifugation at 3000g, and yeast membrane were collected by ultracentrifugation at 100,000g for 30 min at 4 °C. Prepared membranes were resuspended in the membrane buffer [50 mM HEPES pH 7.5, 120 mM NaCl, 20% (v/v) glycerol and EDTA-free protease inhibitor cocktail], and snap-frozen in liquid nitrogen and stored at 80 °C until use. The expression level was evaluated by the single point ligand binding assay using [3H] pyrilamine. Membrane containing 10 lg protein was suspended in the assay buffer [50 mM Tris–HCl pH 7.5, 5 mM MgCl2] and incubated with 40 nM [3H] pyrilamine in a total assay volume of 200 ll for 1 h at 25 °C. Nonspecific binding was determined in the presence of 1000-times excess amount of unlabeled pyrilamine. Membranes were trapped on Whatman GF/B filters pre-soaked in 0.3% polyethylenimine, and unbound radioligands were washed with 9 ml of water. The retained radioactivity was measured on an LCS-5100 liquid scintillation counter (ALOKA) in a Clearzol I scintillation liquid (Nakarai, Japan).
containing 2.5% (v/v) DMSO at 30 °C. Specific binding of [3H] pyrilamine to the receptor was about 5-fold higher when expressed without a-factor signal sequence than with a-factor signal sequence (Fig. 2a). Next, we cultured cells in different conditions (pH of BMMY medium, temperature, and the incubation time) to optimize the culture condition for the overexpression of H1R-T4L. Transformant was inoculated into BMGY medium pH 6.0 at 30 °C, then the cells were transferred to 5 ml of BMMY medium pH 6.0, 7.0 and 8.0 containing 2.5% (v/v) DMSO, and cultured at 30 or 20 °C. Cells were harvested within 20 hours (at 30 °C) or 40 h (at 20 °C) after induction, and the expression levels were examined by ligand binding assay using [3H]-pyrilamine. The highest expression level was observed when cells were cultured at 30 °C in pH 6.0 BMMY medium for 20 h (Fig. 2b).
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3.3. Optimization of the expression condition Because some GPCRs showed higher expression level when expressed with the yeast a-factor signal sequence [24–27], we first examined the effect of the signal sequence on the expression system of H1R-T4L. For this purpose, we generated the expression vector carrying H1R-T4L gene with a-factor signal sequence in its Nterminus (aFac-H1R-T4L), and compared the expression levels between H1R-T4L and aFac-H1R-T4L. P. pastoris transformants harboring the H1R-T4L gene or the aFac-H1R-T4L gene was cultured at small-scale and induced expression in BMMY medium pH 7.0
Fig. 2. Optimization of the culture condition. (a) Specific binding of [3H] pyrilamine to H1R-T4L expressed with or without N-terminal a-factor signal sequence. (b) Specific binding of [3H] pyrilamine to H1R-T4L expressed in different conditions. (c) FSEC profile of H1R-T4L expressed in the presence or absence of doxepin. Membranes was solubilized in the buffer containing 1% DDM/0.2% CHS at final concentration of 1.2 mg/ml, and analyzed by the GFP fluorescence with Superdex 200 5/150 (GE healthcare). Vo represents void volume.
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Finally, we examined the stabilizing effect of the antagonist (doxepin) addition in BMMY medium. Cells were cultured in the presence or absence of 20 lM doxepin. Membrane was prepared as in Section 5.1 and subject to FSEC. Fig. 2c showed that the population of the receptor eluted in the void fraction decreased when cells were cultured with 20 lM doxepin.
cleaved off by the treatment with His-tagged TEV protease [20] (expressed and purified in house). Purified H1R-T4L was separated by Ni-Sepharose affinity chromatography from His-tagged TEV protease and the cleaved His8-tagged GFP.
3.4. Large-scale expression of H1R-T4L
Cells were harvested by centrifugation at 5000g for 20 min at 4 °C. From 8 L culture, 100 g of wet cells were typically obtained. The cells were suspended in breaking buffer [50 mM HEPES, pH 7.5, 120 mM NaCl, 5%(v/v) glycerol, 2 mM EDTA and EDTA-free protein inhibitor cocktail (Roche)]. The suspended cells were mixed with 0.5 mm glass beads and disrupted by shaking at 350 rpm for 2 h at 4 °C. The breakage efficiency of cells was confirmed to be more than 90% using a phase contrast microscope. Undisrupted cells and cell debris were separated by centrifugation at 3000g for 20 min, and then yeast membrane were collected by ultracentrifugation at 100,000g for 30 min at 4 °C.
We expressed H1R-T4L without the yeast a-factor signal sequence as below. Cells from a single colony was inoculated into BMGY medium at pH 6.0 in 500 ml baffled flask with shaking at 250 rpm at 30 °C until an OD600 of 2–6 was reached. The cells were harvested by centrifugation. Then, the cell pellet was resuspended to an OD600 of 1.0 in 1 L of BMMY medium at pH 6.0 in 2.5 L Tunair flask (Sigma) containing 2.5% (v/v) DMSO and 20 lM doxepin at 30 °C for 20 h until the OD600 reached 13. Cells were harvested by centrifugation, and stored at 80 °C. 4. Measurement of the binding activity of H1R-T4L
5.1. Membrane preparation
5.2. Membrane wash
Membrane containing 5 lg protein was suspended in PBS buffer pH 7.4 [1.8 mM KH2PO4 , 8.1 mM Na2HPO4, 27 mM KCl, 138 mM NaCl] and incubated with increasing concentrations of [3H] pyrilamine (from 0.15 to 40 nM) in a total assay volume of 200 ll for 1 h at 25 °C. Nonspecific binding was determined in the presence of 1000-times excess amount of unlabeled pyrilamine. Membranes were trapped on Whatman GF/B filters pre-soaked in 0.3% polyethylenimine, and unbound radioligands were washed with 9 ml of the PBS buffer. The retained radioactivity was measured on an LCS-5100 liquid scintillation counter in a Clearzol I scintillation liquid. Data were analyzed by non-linear curve-fitting with a rectangular hyperbola function using the Prism 4.0 software to determine dissociation constant (Kd). The saturation binding of pyrilamine is shown in Fig. 3. As reported [16], the expressed H1R-T4L showed similar binding affinity for pyrilamine and for histamine as the wild type receptor expressed in Sf9 and COS-7 cells [28].
The membrane was suspended in 400 ml of low salt buffer [10 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl and EDTA-free protease inhibitor cocktail] and homogenized by dounce. The membrane was collected by ultracentrifugation at 100,000g for 30 min at 4 °C. These procedures were repeated 1–3 times. Then, the membrane was suspended in 400 ml of high salt buffer [10 mM HEPES, pH 7.5, 1 M NaCl, 10 mM MgCl2, 20 mM KCl and EDTA-free protease inhibitor cocktail] and homogenized by dounce. The membrane was collected by ultracentrifugation at 100,000g for 30 min at 4 °C. These procedures were repeated four times as was done in the structural studies of other GPCRs [7,9,10]. Washed membranes were resuspended in a buffer containing 50 mM HEPES pH 7.5, 120 mM NaCl, 20% (v/v) glycerol and EDTA-free protease inhibitor cocktail, and frozen in liquid nitrogen and stored at 80 °C until use. The concentration of membrane proteins was estimated using the bicinchoninic acid method (Pierce). From 8 L culture, 1.2 g of membrane proteins were collected.
5. Purification of H1R-T4L
5.3. Solubilization
H1R-T4L was tagged with His8-tag useful for efficient purification. Solubilized H1R-T4L was separated from the yeast membrane proteins by Talon metal affinity chromatography. After removal of imidazole, H1R-T4L was concentrated by Ni-sepharose affinity chromatography. This step was important to reduce the total amount of DDM in the protein solution, because n-dodecyl-b-Dmaltopyranoside (DDM) destabilizes the cubic phase at high concentrations [29]. His8-tagged GFP region of H1R-T4L were then
Membrane suspension (80 ml) was incubated for 30 min at 4 °C with 5 mM doxepin, 2 mg/ml iodoacetamide and EDTA-free protease inhibitor cocktail (Roche). Equal volume of the solubilization buffer [100 mM HEPES pH 7.5, 500 mM NaCl, 1% (w/v) n-dodecyl-b-D-maltopyranoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma), 20% (v/v) glycerol] was added to the membrane suspension, and stirred gently for 90 min at 4 °C. 5.4. Talon metal affinity chromatography
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[ 3 H] pyrilamine [nM] Fig. 3. Saturation binding of [3H] pyrilamine to P. pastoris membrane. The dissociation constant (Kd) obtained by non-linear curve fitting was 4.9 ± 0.3 nM (the average of three independent experiment ± standard error) [16].
After solubilization, unsolubilized materials were separated by ultracentrifugation at 100,000g for 40 min. The supernatant was mixed with 20 ml of TALON IMAC resin (Clontech) and stirred gently at 4 °C overnight. The resin was washed by 200 ml of wash buffer 1 [50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 10 mM MgCl2, 8 mM ATP, 100 lM doxepin and 30 mM imidazole] and 100 ml of wash buffer 2 [50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 100 lM doxepin and 30 mM imidazole]. The receptor was eluted with 100 ml of elution buffer [50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 500 lM doxepin and 200 mM imidazole]. Each fraction was checked by SDS–PAGE, and fractions containing H1T4L were pooled and concentrated to 2.5 ml with a 100 kDa molecular weight cut-off AmiconUltra (Millipore). The concentrated
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Fig. 4. Evaluation of the purified H1R-T4L. (a) Coomassie brilliant blue-stained SDS–PAGE gel of H1R-T4L. (1) Molecular weight marker. (2) Elution of the Talon metal affinity chromatography (Section 5.4). (3) Sample after TEV cleavage (Section 5.6). (4) Elution after removal of His8-tagged GFP and His-tagged TEV by Ni-Sepharose high performance resin (Section 5.6). (b) Chromatogram of gel filtration by Superdex 200 10/300. Arrow indicates the peak of purified H1R-T4L.
receptor solution was applied on PD-10 column (GE healthcare) to remove imidazole. H1-T4L was eluted in 3.5 ml of elution buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS and 100 lM doxepin). 5.5. Ni-sepharose affinity chromatography The eluted protein was mixed with 24 ml of Ni-Sepharose high performance resin (GE healthcare). The resin was washed with 50 ml of wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, 500 lM doxepin and 20 mM imidazole). The sample was eluted with 40 ml of elution buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, 1 mM doxepin and 500 mM imidazole). Imidazole was removed using PD-10 column (GE healthcare). Each fraction was checked by SDS–PAGE, and fractions containing H1-T4L were pooled and concentrated to 2.5 ml with a 100 kDa molecular weight cut-off AmiconUltra (Millipore). Imidazole was removed using PD-10 column (GE healthcare). 5.6. Cleavage of GFP-His8 region 3.5 ml of receptor solution was mixed with 500 ll of Histagged TEV protease [20] (10 mg/ml) and incubated for 15 h at 4 °C. The molecular ratio of the receptor and the protease was 1:1.5. The mixture was then applied on 2–4 ml of Ni-Sepharose high performance resin, and the flow-through was collected (Fraction 1). The resin was washed with one column volume of the buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, and 1 mM doxepin) and the flow-through was collected (Fraction 2). Fractions 1 and 2 were pooled and the receptor concentration was checked using the bicinchoninic acid method. The purity was estimated to be more than 90% by SDS– PAGE (Fig. 4a). The monodispersity was monitored by size-exclusion chromatography using Superdex 200 (GE healthcare) (Fig. 4b). The sample was concentrated to 30 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator and subject to crystallization trials. 6. Conclusion The expression host of GPCRs for structural studies has been limited to insect cells. H1R was the first example whose structure was determined from the receptor expressed in P. pastoris. Here, we mentioned the methods for the production of H1R suitable for the structure determination using the yeast system. By the several optimization steps, the expression level of H1R was improved. Using this yeast expression system, we succeeded to obtain
2 mg of purified H1R-T4L from 8 L yeast culture. This yield is sufficient for the structural study because 1 mg of the purified protein was generally required for crystallization trials [30]. Actually, using the receptor prepared by the methods mentioned above, we succeeded to crystallize H1R in lipidic cubic phase and determined the structure at 3.1 Å resolution [16]. Acknowledgments This work was supported by the ERATO Human Receptor Crystallography Project from the Japan Science and Technology Agency and by the Targeted Proteins Research Program of MEXT (S.I.), Japan. The work was also partly funded by Grant-in-Aid for challenging Exploratory Research (T.S.) and Grant-in-Aid for Scientific Research (B) (T.S.), Grant-in-Aid for Young Scientists (B) (M.S.) the Mochida Memorial Foundation for Medical and Pharmaceutical Research (T.S., T.K.), Takeda Scientific Foundation (M.S.) and the Sumitomo Foundation (T.K.). References [1] R. Fredriksson, M.C. Lagerstrom, L.G. Lundin, H.B. Schioth, Mol. Pharmacol. 63 (2003) 1256–1272. [2] K. Palczewski, T. Kumasaka, T. Hori, C.A. Behnke, H. Motoshima, B.A. Fox, I. Le Trong, D.C. Teller, T. Okada, R.E. Stenkamp, M. Yamamoto, M. Miyano, Science 289 (2000) 739–745. [3] M. Murakami, T. Kouyama, Nature 453 (2008) 363–367. [4] T. Shimamura, K. Hiraki, N. Takahashi, T. Hori, H. Ago, K. Masuda, K. Takio, M. Ishiguro, M. Miyano, J. Biol. Chem. 283 (2008) 17753–17756. [5] K. Lundstrom, R. Wagner, C. Reinhart, A. Desmyter, N. Cherouati, T. Magnin, G. Zeder-Lutz, M. Courtot, C. Prual, N. Andre, G. Hassaine, H. Michel, C. Cambillau, F. Pattus, J. Struct. Funct. Genomics 7 (2006) 77–91. [6] S.G. Rasmussen, H.J. Choi, D.M. Rosenbaum, T.S. Kobilka, F.S. Thian, P.C. Edwards, M. Burghammer, V.R. Ratnala, R. Sanishvili, R.F. Fischetti, G.F. Schertler, W.I. Weis, B.K. Kobilka, Nature 450 (2007) 383–387. [7] V. Cherezov, D.M. Rosenbaum, M.A. Hanson, S.G. Rasmussen, F.S. Thian, T.S. Kobilka, H.J. Choi, P. Kuhn, W.I. Weis, B.K. Kobilka, R.C. Stevens, Science 318 (2007) 1258–1265. [8] V.P. Jaakola, M.T. Griffith, M.A. Hanson, V. Cherezov, E.Y. Chien, J.R. Lane, A.P. Ijzerman, R.C. Stevens, Science 322 (2008) 1211–1217. [9] B. Wu, E.Y. Chien, C.D. Mol, G. Fenalti, W. Liu, V. Katritch, R. Abagyan, A. Brooun, P. Wells, F.C. Bi, D.J. Hamel, P. Kuhn, T.M. Handel, V. Cherezov, R.C. Stevens, Science 330 (2010) 1066–1071. [10] E.Y. Chien, W. Liu, Q. Zhao, V. Katritch, G.W. Han, M.A. Hanson, L. Shi, A.H. Newman, J.A. Javitch, V. Cherezov, R.C. Stevens, Science 330 (2010) 1091– 1095. [11] T. Warne, M.J. Serrano-Vega, J.G. Baker, R. Moukhametzianov, P.C. Edwards, R. Henderson, A.G. Leslie, C.G. Tate, G.F. Schertler, Nature 454 (2008) 486–491. [12] D.M. Rosenbaum, V. Cherezov, M.A. Hanson, S.G. Rasmussen, F.S. Thian, T.S. Kobilka, H.J. Choi, X.J. Yao, W.I. Weis, R.C. Stevens, B.K. Kobilka, Science 318 (2007) 1266–1273. [13] J.C. Schwartz, J.M. Arrang, M. Garbarg, H. Pollard, M. Ruat, Physiol. Rev. 71 (1991) 1–51. [14] S.J. Hill, Pharmacol. Rev. 42 (1990) 45–83. [15] S.J. Hill, C.R. Ganellin, H. Timmerman, J.C. Schwartz, N.P. Shankley, J.M. Young, W. Schunack, R. Levi, H.L. Haas, Pharmacol. Rev. 49 (1997) 253–278.
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[16] T. Shimamura, M. Shiroishi, S. Weyand, H. Tsujimoto, G. Winter, V. Katritch, R. Abagyan, V. Cherezov, W. Liu, G.W. Han, T. Kobayashi, R.C. Stevens, S. Iwata, Nature 475 (2011) 65–70. [17] T. Warne, J. Chirnside, G.F. Schertler, Biochim. Biophys. Acta 1610 (2003) 133– 140. [18] T. Yurugi-Kobayashi, H. Asada, M. Shiroishi, T. Shimamura, S. Funamoto, N. Katsuta, K. Ito, T. Sugawara, N. Tokuda, H. Tsujimoto, T. Murata, N. Nomura, K. Haga, T. Haga, S. Iwata, T. Kobayashi, Biochem. Biophys. Res. Commun. 380 (2009) 271–276. [19] T. Kawate, E. Gouaux, Structure 14 (2006) 673–681. [20] S. Newstead, H. Kim, G. Von Heijne, S. Iwata, D. Drew, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 13936–13941. [21] K. Ito, T. Sugawara, M. Shiroishi, N. Tokuda, A. Kurokawa, T. Misaka, H. Makyio, T. Yurugi-Kobayashi, T. Shimamura, N. Nomura, T. Murata, K. Abe, S. Iwata, T. Kobayashi, Biochem. Biophys. Res. Commun. 371 (2008) 841–845.
[22] J. Kota, C.F. Gilstring, P.O. Ljungdahl, J. Cell Biol. 176 (2007) 617–628. [23] N. Andre, N. Cherouati, C. Prual, T. Steffan, G. Zeder-Lutz, T. Magnin, F. Pattus, H. Michel, R. Wagner, C. Reinhart, Protein Science : A Publication of the Protein Society 15 (2006) 1115–1126. [24] H.M. Weiss, W. Haase, H. Michel, H. Reilander, FEBS Lett. 377 (1995) 451–456. [25] F. Talmont, S. Sidobre, P. Demange, A. Milon, L.J. Emorine, FEBS Lett. 394 (1996) 268–272. [26] B. Andersen, R.C. Stevens, Prot. Expr. Purif. 13 (1998) 111–119. [27] S. Grunewald, W. Haase, E. Molsberger, H. Michel, H. Reilander, Receptors Channels 10 (2004) 37–50. [28] V.R. Ratnala, H.G. Swarts, J. Vanoostrum, R. Leurs, H.J. Degroot, R.A. Bakker, W.J. Degrip, Eur. J. Biochem. 271 (2004) 2636–2646. [29] Y. Misquitta, M. Caffrey, Biophys. J. 85 (2003) 3084–3096. [30] V. Sarramegna, F. Talmont, P. Demange, A. Milon, Cell. Mol. Life Sci. 60 (2003) 1529–1546.
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Production of the stable human histamine H1 receptor in Pichia pastoris for structural determination Mitsunori Shiroishi a,b, Takuya Kobayashi a,c, Satoshi Ogasawara c, Hirokazu Tsujimoto a,c, Chiyo Ikeda-Suno a,c, So Iwata a,c,d,e, Tatsuro Shimamura a,c,⇑ a
Human Receptor Crystallography Project, ERATO, Japan Science and Technology Agency, Yoshidakonoe-cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan d Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK e Diamond Light Source, Harwell Science and Innovation Campus, Chilton, Didcot, Oxfordshire OX11 0DE, UK b c
a r t i c l e
i n f o
Article history: Available online 27 August 2011 Keywords: GPCR Expression Pichia pastoris Yeast Structure Membrane protein
a b s t r a c t G-protein coupled receptors (GPCRs) play essential roles in regulation of many physiological processes and are one of the major targets of pharmaceutical drugs. The 3D structure can provide important information for the understanding of GPCR function and the design of new drugs. However, the success of structure determination relies largely on the production of recombinant GPCRs, because the expression levels of GPCRs are very low in native tissues except rhodopsin. All non-rhodopsin GPCRs whose structures were determined so far were expressed in insect cells and the availability of other hosts was unknown. Recently, we succeeded to determine the structure of human histamine H1 receptor (H1R) expressed in Pichia pastoris. Here, we report the expression and purification procedures of recombinant H1R used in the structural determination. The receptor was designed to possess a N-terminal 19-residue deletion and a replacement of the third cytoplasmic loop with T4-lysozyme. The receptor was verified to show similar binding activities with the receptor expressed in other hosts. The receptor was purified by the immobilized metal ion affinity chromatography and used for the crystallographic study that resulted in the successful structure determination. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction G-protein coupled receptors (GPCRs) are one of the largest family of membrane proteins and more than 800 GPCRs are identified in human [1]. GPCRs play essential roles in the signal transduction pathway by receiving extracellular signals and activating G-proteins to relay the signal inside the cell. Reflecting the pivotal functions, GPCRs constitute 50% of all drug targets. Until 2007, only rhodopsin structures [2–4] were available to the structural modeling of other GPCRs for effective development of new drugs. Rhodopsin is the exceptional GPCR because the receptor is stabilized by covalently bound retinal, and can be purified abundantly from retina for the structure determination. In contrast, most GPCRs are expressed at low level and difficult to purify from the native tissues. Therefore, for the GPCR structural studies, it is essential to establish the overexpression system of stable receptors. However, recombinant production of functional GPCRs is known to be ⇑ Corresponding author at: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshidakonoe-cho, Sakyo-Ku, Kyoto 606-8501, Japan. Fax: +81 75 753 4660. E-mail address: [email protected] (T. Shimamura). 1046-2023/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2011.08.015
difficult partly due to their conformational flexibility [5]. Five non-rhodopsin GPCR structures have been determined so far using the stabilized receptors by protein engineering. They are human b2-adrenergic receptor (b2AR) [6,7], human adenosine A2A receptor (A2AAR) [8], human CXCR4 chemokine receptor [9], human dopamine D3 receptor [10] and turkey b1-adrenergic receptor (b1AR) [11]. The structure of b1AR was determined by the vapor diffusion crystallization method using the thermostabilized multiple mutant. As for b2AR, one structure was determined as a Fab complex by the vapor diffusion crystallization method [6]. The other structure was determined from the crystals formed in lipidic cubic phase [7]. In this case, the flexible third cytoplasmic loop was replaced with T4-lysozyme (T4L) [12]. The latter strategy was successfully applied to the structural studies of other human GPCRs [8–10]. Though the stabilization strategies were different, however, these receptors were all overexpressed in insect cells and the availability of other hosts for the GPCR structural study was unknown. Human histamine H1 receptor (H1R) is involved in allergic responses and the target of the antihistamine drugs [13–15]. Recently, we succeeded to determine the structure of H1R at 3.1 Å resolution using lipidic cubic phase crystallization method [16].
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The receptor construct contained a replacement of the third cytoplasmic loop with T4L (H1R-T4L) and was expressed in Pichia pastoris. Here we describe the method how we established the overexpression system of H1R in yeast and purification system of the receptor used for the structure determination [16].
the T4L insertion site and the deletion length in N and C terminal regions. However, it is sometimes necessary to prepare and screen several constructs as were done in the structural studies of other GPCRs [7,9,10,12]. 3. Expression of H1R-T4L in P. pastoris
2. Design of the H1R-T4L construct Fig. 1a shows the construct of H1R-T4L used for the expression in P. pastoris. The construct consists of H1R with a N-terminal 19residue deletion and a replacement of the third cytoplasmic loop by T4L, tobacco etch virus (TEV) protease cleavage site, green fluorescent protein (GFP) and octa-histidine (His8) tag. This construct was designed based on the structural information of three nonrhodopsin GPCRs (b1AR [11], b2AR [7] and A2AAR [8]) available at that time. In these structures, N- and C-terminal regions were disordered (Fig. 1b and c), suggesting that these regions are flexible and could affect badly for the expression of GPCRs [17]. Therefore, we truncated the N-terminal 19 residues (Met1-Lys19) of H1R. It should be noted that the truncated region contains two N-linked glycosylation sites (Asn5 and Asn18) that can be obstacles to crystal packing [18]. The C-terminal region of H1R was proposed to be very short after the helix VIII by the alignment (Fig. 1c) and we did not delete any residues at C-terminal region. We replaced the third cytoplasmic loop between 5th and 6th transmembrane helices with T4L at the corresponding site to b2AR that is an aminergic receptor similarly with H1R. GFP was fused to the receptor for the evaluation of monodispersity by fluorescence size exclusion chromatography (FSEC) [19]. His8-tag was tagged for the affinity purification at the C-terminal of GFP. GFP and His8-tag were removed at the final step of the purification procedure by TEV protease cleavage. Consequently, H1R-T4L showed better expression level, binding activity to pyrilamine, and FSEC profile than the full-length H1R when expressed in Saccharomyces cerevisiae, suggesting that the stability of the receptor was increased by these modifications (Shiroishi, Unpublished data). The construct was used for the structural determination without further optimizing
In this study, we used two yeast species, S. cerevisiae and P. pastoris. P. pastoris was used for the expression of H1R-T4L because it generates large biomass in fermentor cultures [5]. S. cerevisiae was used for the construction of the H1R-T4L gene because the plasmid was easily and quickly assembled by yeast homologous recombination of PCR fragments [20,21]. 3.1. Construction of the H1R-T4L gene in S. cerevisiae The template DNA encoding full-length H1R was codon-optimized to yeast and synthesized. Three PCR fragments encoding Thr20-Cys221 of H1R, Asn2-Tyr161 of T4L and Leu405-Ser487 of H1R, respectively, were amplified using appropriate primers and templates by the standard PCR using KOD plus DNA polymerase (TOYOBO, Japan). A 30–40 ng of SmaI-linearized plasmid pDDGFP2 [20] and 3 ll of the PCR reaction mixtures were co-transformed into S. cerevisiae strain FGY217 [20,22]. Transformants harboring the plasmid encoding H1R-T4L were selected on–Ura–agar plates [0.192% (w/v) yeast synthetic drop-out media without Ura (Sigma), 0.67% (w/v) yeast nitrogen base without amino acids (BD), 2% (w/v) agar and 2% (w/v) glucose]. The S. cerevisiae transformant was cultured in 5 ml of–Ura medium [0.192% (w/v) yeast synthetic dropout media without Ura, 0.671% (w/v) yeast nitrogen base without amino acids and 2% (w/v) glucose] at 30 °C for 22–24 h. The generated plasmid encoding H1R-T4L was isolated from S. cerevisiae with the Miniprep Kit (Qiagen) by disrupting cells with 0.5 mm glass beads. The plasmid was amplified in Escherichia coli strain DH5a, and the DNA sequence for H1R-T4L was verified by DNA sequencing. Coding regions of H1R-T4L followed by TEV protease recognition site, GFP and His8-tag were amplified by PCR using a
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Fig. 1. Design of H1R-T4L construct. (a) Schematic representation of H1R-T4L construct. Residues 222-404 of H1R were replaced by T4L. The H1R-T4L is connected to His8tagged GFP by the TEV protease recognition sequence. (b) Sequence alignment of N-terminal region between H1R, b2AR and A2AAR. Conserved residue was shown in red. Disordered residues in b2AR and A2AAR structures are shown in grey. The truncated residues of H1R were shown in green. (c) Sequence alignment of C-terminal region. (d) Sequence alignment around T4L insertion region.
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forward primer containing a BamHI site (50 -CTA GAA CTA GTG GAT CCA CCA TG-30 ) and a reverse primer containing an EcoRI site (50 GCT TGA TAT CGA ATT CCT GCA GTT AAT G-30 ). The PCR products were digested with BamHI and EcoRI, and subcloned into the pPIC9K vector. 3.2. Integration of the H1R-T4L gene into the genome of P. pastoris The PmeI linearized pPIC9K vector prepared in Section 3.1 was transformed into the P. pastoris SMD1163 strain by electroporation (2000 V, 25 lF, and 600 X) using a Gene Pulser I (Bio-Rad). Then, we selected the clone with the highest expression level and used for the large-scale expression. Clone selection was performed on the MD-agar plate [1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 2% (w/v) glucose and 1.5% (w/v) agar], followed by YPD-agar plate containing 0.1 mg/ml geneticine. To examine the expression level of H1R-T4L, the clones were cultured at small-scale as follows. A single colony of P. pastoris transformant on the YPD plate with 0.1 mg/ml geneticine was inoculated into 5 ml of BMGY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 1% (w/v) glycerol, 0.1 M phosphate buffer at pH 6.0] at 30 °C with shaking at 250 rpm until an OD600 of 2–6 was reached. The cells were harvested by centrifugation. To induce expression, the cell pellet was resuspended to an OD600 of 1.0 in 5 ml of BMMY medium [1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) yeast nitrogen base without amino acids, 0.00004% (w/v) biotin, 0.5% (v/v) methanol, 0.1 M phosphate buffer at pH 7.0] containing 2.5% (v/v) DMSO at 30 °C. The addition of DMSO has been known to improve the expression level of GPCRs in P. pastoris [23]. Cells were harvested within 20–22 h after induction. Yeast cells were suspended in 500 ll of the breaking buffer [50 mM HEPES pH 7.5, 120 mM NaCl, 5%(v/v) glycerol, 2 mM EDTA and EDTA-free protein inhibitor cocktail (Roche)]. Cells were disrupted with 500 ll of 0.5 mm glass beads by vortexing at 2500 rpm in 2 ml centrifuge tube. Undisrupted cells and cell debris were separated by centrifugation at 3000g, and yeast membrane were collected by ultracentrifugation at 100,000g for 30 min at 4 °C. Prepared membranes were resuspended in the membrane buffer [50 mM HEPES pH 7.5, 120 mM NaCl, 20% (v/v) glycerol and EDTA-free protease inhibitor cocktail], and snap-frozen in liquid nitrogen and stored at 80 °C until use. The expression level was evaluated by the single point ligand binding assay using [3H] pyrilamine. Membrane containing 10 lg protein was suspended in the assay buffer [50 mM Tris–HCl pH 7.5, 5 mM MgCl2] and incubated with 40 nM [3H] pyrilamine in a total assay volume of 200 ll for 1 h at 25 °C. Nonspecific binding was determined in the presence of 1000-times excess amount of unlabeled pyrilamine. Membranes were trapped on Whatman GF/B filters pre-soaked in 0.3% polyethylenimine, and unbound radioligands were washed with 9 ml of water. The retained radioactivity was measured on an LCS-5100 liquid scintillation counter (ALOKA) in a Clearzol I scintillation liquid (Nakarai, Japan).
containing 2.5% (v/v) DMSO at 30 °C. Specific binding of [3H] pyrilamine to the receptor was about 5-fold higher when expressed without a-factor signal sequence than with a-factor signal sequence (Fig. 2a). Next, we cultured cells in different conditions (pH of BMMY medium, temperature, and the incubation time) to optimize the culture condition for the overexpression of H1R-T4L. Transformant was inoculated into BMGY medium pH 6.0 at 30 °C, then the cells were transferred to 5 ml of BMMY medium pH 6.0, 7.0 and 8.0 containing 2.5% (v/v) DMSO, and cultured at 30 or 20 °C. Cells were harvested within 20 hours (at 30 °C) or 40 h (at 20 °C) after induction, and the expression levels were examined by ligand binding assay using [3H]-pyrilamine. The highest expression level was observed when cells were cultured at 30 °C in pH 6.0 BMMY medium for 20 h (Fig. 2b).
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3.3. Optimization of the expression condition Because some GPCRs showed higher expression level when expressed with the yeast a-factor signal sequence [24–27], we first examined the effect of the signal sequence on the expression system of H1R-T4L. For this purpose, we generated the expression vector carrying H1R-T4L gene with a-factor signal sequence in its Nterminus (aFac-H1R-T4L), and compared the expression levels between H1R-T4L and aFac-H1R-T4L. P. pastoris transformants harboring the H1R-T4L gene or the aFac-H1R-T4L gene was cultured at small-scale and induced expression in BMMY medium pH 7.0
Fig. 2. Optimization of the culture condition. (a) Specific binding of [3H] pyrilamine to H1R-T4L expressed with or without N-terminal a-factor signal sequence. (b) Specific binding of [3H] pyrilamine to H1R-T4L expressed in different conditions. (c) FSEC profile of H1R-T4L expressed in the presence or absence of doxepin. Membranes was solubilized in the buffer containing 1% DDM/0.2% CHS at final concentration of 1.2 mg/ml, and analyzed by the GFP fluorescence with Superdex 200 5/150 (GE healthcare). Vo represents void volume.
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Finally, we examined the stabilizing effect of the antagonist (doxepin) addition in BMMY medium. Cells were cultured in the presence or absence of 20 lM doxepin. Membrane was prepared as in Section 5.1 and subject to FSEC. Fig. 2c showed that the population of the receptor eluted in the void fraction decreased when cells were cultured with 20 lM doxepin.
cleaved off by the treatment with His-tagged TEV protease [20] (expressed and purified in house). Purified H1R-T4L was separated by Ni-Sepharose affinity chromatography from His-tagged TEV protease and the cleaved His8-tagged GFP.
3.4. Large-scale expression of H1R-T4L
Cells were harvested by centrifugation at 5000g for 20 min at 4 °C. From 8 L culture, 100 g of wet cells were typically obtained. The cells were suspended in breaking buffer [50 mM HEPES, pH 7.5, 120 mM NaCl, 5%(v/v) glycerol, 2 mM EDTA and EDTA-free protein inhibitor cocktail (Roche)]. The suspended cells were mixed with 0.5 mm glass beads and disrupted by shaking at 350 rpm for 2 h at 4 °C. The breakage efficiency of cells was confirmed to be more than 90% using a phase contrast microscope. Undisrupted cells and cell debris were separated by centrifugation at 3000g for 20 min, and then yeast membrane were collected by ultracentrifugation at 100,000g for 30 min at 4 °C.
We expressed H1R-T4L without the yeast a-factor signal sequence as below. Cells from a single colony was inoculated into BMGY medium at pH 6.0 in 500 ml baffled flask with shaking at 250 rpm at 30 °C until an OD600 of 2–6 was reached. The cells were harvested by centrifugation. Then, the cell pellet was resuspended to an OD600 of 1.0 in 1 L of BMMY medium at pH 6.0 in 2.5 L Tunair flask (Sigma) containing 2.5% (v/v) DMSO and 20 lM doxepin at 30 °C for 20 h until the OD600 reached 13. Cells were harvested by centrifugation, and stored at 80 °C. 4. Measurement of the binding activity of H1R-T4L
5.1. Membrane preparation
5.2. Membrane wash
Membrane containing 5 lg protein was suspended in PBS buffer pH 7.4 [1.8 mM KH2PO4 , 8.1 mM Na2HPO4, 27 mM KCl, 138 mM NaCl] and incubated with increasing concentrations of [3H] pyrilamine (from 0.15 to 40 nM) in a total assay volume of 200 ll for 1 h at 25 °C. Nonspecific binding was determined in the presence of 1000-times excess amount of unlabeled pyrilamine. Membranes were trapped on Whatman GF/B filters pre-soaked in 0.3% polyethylenimine, and unbound radioligands were washed with 9 ml of the PBS buffer. The retained radioactivity was measured on an LCS-5100 liquid scintillation counter in a Clearzol I scintillation liquid. Data were analyzed by non-linear curve-fitting with a rectangular hyperbola function using the Prism 4.0 software to determine dissociation constant (Kd). The saturation binding of pyrilamine is shown in Fig. 3. As reported [16], the expressed H1R-T4L showed similar binding affinity for pyrilamine and for histamine as the wild type receptor expressed in Sf9 and COS-7 cells [28].
The membrane was suspended in 400 ml of low salt buffer [10 mM HEPES, pH 7.5, 10 mM MgCl2, 20 mM KCl and EDTA-free protease inhibitor cocktail] and homogenized by dounce. The membrane was collected by ultracentrifugation at 100,000g for 30 min at 4 °C. These procedures were repeated 1–3 times. Then, the membrane was suspended in 400 ml of high salt buffer [10 mM HEPES, pH 7.5, 1 M NaCl, 10 mM MgCl2, 20 mM KCl and EDTA-free protease inhibitor cocktail] and homogenized by dounce. The membrane was collected by ultracentrifugation at 100,000g for 30 min at 4 °C. These procedures were repeated four times as was done in the structural studies of other GPCRs [7,9,10]. Washed membranes were resuspended in a buffer containing 50 mM HEPES pH 7.5, 120 mM NaCl, 20% (v/v) glycerol and EDTA-free protease inhibitor cocktail, and frozen in liquid nitrogen and stored at 80 °C until use. The concentration of membrane proteins was estimated using the bicinchoninic acid method (Pierce). From 8 L culture, 1.2 g of membrane proteins were collected.
5. Purification of H1R-T4L
5.3. Solubilization
H1R-T4L was tagged with His8-tag useful for efficient purification. Solubilized H1R-T4L was separated from the yeast membrane proteins by Talon metal affinity chromatography. After removal of imidazole, H1R-T4L was concentrated by Ni-sepharose affinity chromatography. This step was important to reduce the total amount of DDM in the protein solution, because n-dodecyl-b-Dmaltopyranoside (DDM) destabilizes the cubic phase at high concentrations [29]. His8-tagged GFP region of H1R-T4L were then
Membrane suspension (80 ml) was incubated for 30 min at 4 °C with 5 mM doxepin, 2 mg/ml iodoacetamide and EDTA-free protease inhibitor cocktail (Roche). Equal volume of the solubilization buffer [100 mM HEPES pH 7.5, 500 mM NaCl, 1% (w/v) n-dodecyl-b-D-maltopyranoside (DDM, Anatrace), 0.2% (w/v) cholesteryl hemisuccinate (CHS, Sigma), 20% (v/v) glycerol] was added to the membrane suspension, and stirred gently for 90 min at 4 °C. 5.4. Talon metal affinity chromatography
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[ 3 H] pyrilamine [nM] Fig. 3. Saturation binding of [3H] pyrilamine to P. pastoris membrane. The dissociation constant (Kd) obtained by non-linear curve fitting was 4.9 ± 0.3 nM (the average of three independent experiment ± standard error) [16].
After solubilization, unsolubilized materials were separated by ultracentrifugation at 100,000g for 40 min. The supernatant was mixed with 20 ml of TALON IMAC resin (Clontech) and stirred gently at 4 °C overnight. The resin was washed by 200 ml of wash buffer 1 [50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 10 mM MgCl2, 8 mM ATP, 100 lM doxepin and 30 mM imidazole] and 100 ml of wash buffer 2 [50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 100 lM doxepin and 30 mM imidazole]. The receptor was eluted with 100 ml of elution buffer [50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS, 500 lM doxepin and 200 mM imidazole]. Each fraction was checked by SDS–PAGE, and fractions containing H1T4L were pooled and concentrated to 2.5 ml with a 100 kDa molecular weight cut-off AmiconUltra (Millipore). The concentrated
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Fig. 4. Evaluation of the purified H1R-T4L. (a) Coomassie brilliant blue-stained SDS–PAGE gel of H1R-T4L. (1) Molecular weight marker. (2) Elution of the Talon metal affinity chromatography (Section 5.4). (3) Sample after TEV cleavage (Section 5.6). (4) Elution after removal of His8-tagged GFP and His-tagged TEV by Ni-Sepharose high performance resin (Section 5.6). (b) Chromatogram of gel filtration by Superdex 200 10/300. Arrow indicates the peak of purified H1R-T4L.
receptor solution was applied on PD-10 column (GE healthcare) to remove imidazole. H1-T4L was eluted in 3.5 ml of elution buffer (50 mM Hepes pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% (w/v) DDM, 0.01% (w/v) CHS and 100 lM doxepin). 5.5. Ni-sepharose affinity chromatography The eluted protein was mixed with 24 ml of Ni-Sepharose high performance resin (GE healthcare). The resin was washed with 50 ml of wash buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, 500 lM doxepin and 20 mM imidazole). The sample was eluted with 40 ml of elution buffer (20 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, 1 mM doxepin and 500 mM imidazole). Imidazole was removed using PD-10 column (GE healthcare). Each fraction was checked by SDS–PAGE, and fractions containing H1-T4L were pooled and concentrated to 2.5 ml with a 100 kDa molecular weight cut-off AmiconUltra (Millipore). Imidazole was removed using PD-10 column (GE healthcare). 5.6. Cleavage of GFP-His8 region 3.5 ml of receptor solution was mixed with 500 ll of Histagged TEV protease [20] (10 mg/ml) and incubated for 15 h at 4 °C. The molecular ratio of the receptor and the protease was 1:1.5. The mixture was then applied on 2–4 ml of Ni-Sepharose high performance resin, and the flow-through was collected (Fraction 1). The resin was washed with one column volume of the buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 0.025% (w/v) DDM, 0.005% (w/v) CHS, and 1 mM doxepin) and the flow-through was collected (Fraction 2). Fractions 1 and 2 were pooled and the receptor concentration was checked using the bicinchoninic acid method. The purity was estimated to be more than 90% by SDS– PAGE (Fig. 4a). The monodispersity was monitored by size-exclusion chromatography using Superdex 200 (GE healthcare) (Fig. 4b). The sample was concentrated to 30 mg/ml with a 100 kDa molecular weight cut-off Vivaspin concentrator and subject to crystallization trials. 6. Conclusion The expression host of GPCRs for structural studies has been limited to insect cells. H1R was the first example whose structure was determined from the receptor expressed in P. pastoris. Here, we mentioned the methods for the production of H1R suitable for the structure determination using the yeast system. By the several optimization steps, the expression level of H1R was improved. Using this yeast expression system, we succeeded to obtain
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