Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system
© Nozawa et al; licensee BioMed Central Ltd. 2011
Received: 29 October 2010
Accepted: 11 April 2011
Published: 11 April 2011
Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.
AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE.
Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.
MPs comprise up to 30% of genes in fully sequenced genomes and have critical roles in a variety of biological processes including signal transduction, substrate transport, and energy production [1, 2]. However, functional and structural studies of MPs are far behind that of soluble proteins. One of the major bottlenecks in the study of MPs is the difficulty in obtaining sufficient amounts of homogeneous protein. For instance, it is typically not easy to purify MPs in preparative scale, due to their low abundance in natural sources. Overexpression of recombinant MPs in living cells is often unsuccessful due to the inhibitory effect of high MP concentration on host cell physiology .
Recently, cell-free protein synthesis systems have emerged as a promising tool for MP production [4–6]. In addition to decoupling protein production from the toxic or inhibitory effects on host cell physiology, cell-free systems offer a unique advantage in that protein synthesis can be easily modified by addition of accessory elements, such as detergents and lipids. The addition of detergents and lipids to cell-free systems allows the synthesis of MP/detergent and MP/lipid complexes, respectively, and successful synthesis of functional MPs in this fashion have been reported recently [7–11]. For example, Klammt et al.  demonstrated that a G protein-coupled receptor, ETB, can be synthesized in a soluble form using an Escherichia coli-based cell-free system supplemented with Brij78, and that the synthesized proteins have ligand binding activity. The ligand binding activity of a human olfactory receptor, hOR17-4, synthesized using a wheat cell-free system in the presence of FC14, has been also reported . Kalmbach et al.  reported that E. coli cell-free synthesized bacteriorhodopsin in the presence of liposomes was active in black lipid membrane mediated photocurrent measurements. Goren and Fox  showed reconstitution of the functional stearoyl Co-A desaturase complex, which consists of three proteins, cytochrome b5, cytochrome b5 reductase, and human stearoyl-CoA desaturase 1 (hSCD1) synthesized by wheat cell-free system in the presence of asolectin liposomes. However, the general versatility of this method is unclear as the above examples focus on specific MPs.
In a previous study, we reported functional synthesis of a phosphate trranslocator in a wheat cell-free synthesis system supplemented with liposomes and formation of lipid/MP complexes . The mechanism for production of functional protein in this method is not clear, but association of synthesized MP with liposomes may be an important step. To better understand this, we tested the timing of liposome addition to the cell-free MP synthesis reaction. We also investigated whether other MPs synthesized by the method also associates with liposomes. Moreover, we tried to purify the synthesized MP as a lipid/MP complex by DGU.
Results and Discussion
Timing of liposome-supplementation to wheat cell-free translation system for synthesis of functional MPs
Cell-free synthesis of MPs in the presence of liposomes
Yield, association rate and solubility of synthesized membrane proteins
Number of TMD
Molecular weight (kDa)
Yield ( μg/150 μl reaction)
Association rates with liposomes (%)
Yield × Association rates ( μg)
Potassium inwardly-rectifying channel
Potassium inwardly-rectifying channel
Potassium inwardly-rectifying channel
Voltage-dependent calcium channel
Voltage-dependent calcium channel
G protein-coupled receptor 37
GABA transporter, GABA
Solute carrier family 6, member 18
Vesicular monoamine transporter
Organic cation transporter
Organic anion transporter
Organic anion transporter
organic anion/cation transporter
Organic anion/cation transporter
Sodium-dependent vitamin transporter
Cationic amino acid transporter
The extent of association of all 40 proteins tested, containing between one and 14 TMDs, were from 43 to 73% and the mean value was 59% (Table 1). The amount of proteins associated with liposomes was calculated from the yield and extent of association for each protein (Figure 3C and Table 1). The mean value of the association yield was 3.4 μg/150 μL reaction. When analyzing association by the number of TMDs, the lowest extent of association was 43% for 1 TMD (Itga1), whereas the mean value of association for proteins having more than 2 TMDs was approximately 60% (Table 1). These results indicate that efficiency of association of proteins having only 1TMD might be lower than that of proteins having more than 2TMD.
Figure 3D shows the relationship between solubility and association rate of tested proteins. Solubility of MPs synthesized using the wheat cell-free system in the absence of liposomes ranged from eight to 61%. The extent of association of these proteins ranged from 40 to 70% regardless of their solubilities. On the other hand, soluble proteins showed more than 80% solubility and their association rates were less than 20%, indicating that the extent of liposome association for soluble proteins is much lower than that of MPs. In comparison to the tested soluble proteins, the MPs examined had a wide variety of sizes, functional roles and topologies, and all of them appeared to be efficiently associated with liposomes during cell-free synthesis. Moreover, there were no significant relationship between the extent of protein association and their respective yields (Figure 3E).
In this experiment, we found that a variety of MPs make complexes with lipids during wheat cell-free synthesis in the presence of liposomes. It is not yet clear whether these MPs are integrated into liposomes or attached to surface of liposomes with their hydrophobic regions. For replying to this question, further experiments for evaluating function and/or structure of each synthesized MP are needed. However, as association of MPs with liposomes prevents aggregation and precipitation of synthesized MPs during synthesis reaction, formation of lipid/MP complex would be an important step for preparing MPs.
Katzen et al.  recently reported that insertion of EmrE into a discoidal membrane scaffold in correctly folded state during cell-free synthesis by analysis of binding activity of its substrate, tetraphenylphosphonium. Moritani et al.  demonstrated that connexin-43 synthesized by an E. coli cell-free system, PURE system containing minimum protein synthesis factors , in the presence of liposomes is directly integrated with a uniform orientation into liposome membrane. The connexin-43 synthesized into liposomes was shown to deliver a hydrophilic and bioactive oligo-peptide to cells through gap junctions . They speculate that liposomes may have chaperone-like function because their system consists of only protein synthesis factors and liposomes . Although it is still not clear any kinds of MPs can receive such a chaperone-like effect from liposomes, the addition of artificial membranes to cell-free reactions appears to be effective in synthesizing membrane-stabilized MPs.
Partial purification of synthesized MPs by density-gradient ultracentrifugation
In this study, we demonstrated that a variety of mammalian MPs are efficiently associated with liposomes during wheat cell-free synthesis supplemented with liposomes. Next, we tried to partially purify the synthesized lipid/MP complexes from the endogenous wheat germ proteins. For this experiment we used P2RX4 as a model protein, because both the yield (6.1 μg) and extent of association (66%) are very close to the mean value for both of these parameters in the full mammalian MP dataset above (5.9 μg and 59%, respectively).
Although fluorescence was seen at the top of the gradient in the soluble GFP control after Accudenz DGU by using a transilluminator (excitation: 400-500 nm) with a filter for removing blue light (Figure 4A), the fluorescence in the top fraction was not detected with a spectrofluorometer using a condition for detection of fluorescence from GFP (excitation 480 nm/emission 510 nm) (Figure 4B). These results indicate that the fluorescence in the top fraction is not attributed to GFP but probably to the liposomes. In comparison to sucrose DGU, Accudenz DGU makes lipid/MP complex recovery easier due to the fact that liposomes float in Accudenz as opposed to sinking as in sucrose. Additionally, Accudenz is a preferred DGU solution because it is non-toxic.
The GFP-fusion P2RX4 is an ideal construct to determine the optimal conditions for Accudenz DGU as the liposome-associated protein can be readily monitored by fluorescence. We tested several centrifugation times for Accudenz DGU. As shown in Figure 4C, the fluorescence in the top fraction gradually increased in proportion centrifugation time. One-h centrifugation appears to be sufficient for lipid/MP complex floatation. The results from Accudenz DGU of two additional MPs, GFP-P2RX1 and GFP-SLC6A18, also showed that the floatation rates of lipid/MP complex plateaus at a one-h centrifugation (Figure 4D).
As shown in Figure 4B, GFP and GFP-P2RX4 could be clearly separated by DGU. The result suggests that lipid/MP complexes can be separated from endogenous proteins derived from the wheat germ extract by Accudenz DGU. Nomura et al. also reported that cytochrome b5 synthesized by the wheat cell-free system in the presence of liposomes was easily purified by simplified discontinuous DGU . In addition, Goren and Fox showed that a MP, hSCD1, synthesized by the wheat cell-free system supplemented with liposomes could be separated from endogenous wheat germ proteins by Accudenz DGU . We next tried to purify P2RX4, without a GFP fusion, by this method. After synthesis supplemented with liposomes, lipid/MP complexes were concentrated by centrifugation and applied to Accudenz DGU. After ultracentrifugation, the top fraction was recovered and applied to SDS-PAGE. As shown in Figure 4E, P2RX4 was detected as a major band by CBB-staining after SDS-PAGE. This result indicates that lipid/MP complexes synthesized by our wheat cell-free system supplemented with liposomes can be partially purified by Accudenz DGU. To test whether proteins purified by this method are functional, we tested the activity of AtPPT1 partially purified as lipid/MP complexes. We detected transport activity of AtPPT1 purified with Accudenz DGU (data not shown). In addition to functional analysis, this partially purified protein could be used for structural analysis. However, some contaminating proteins from wheat germ extracts do float with the lipid/MP complexes. Goren and Fox  reported that, along with other unclassified contaminants, HSP70, elongation factor 1 α and 16.9 kDa heat shock protein were seen in wheat cell-free synthesized and floated lipid/MP complexes. For functional and structural analysis, further purification steps would be required including affinity-tag purification and gel-filtration.
We have developed a production method for lipid/MP complexes using a wheat germ cell-free system supplemented with liposomes. Using this method, a variety of mammalian MPs were efficiently associated with liposomes co-translationally. The resultant lipid/MP complexes are easily separated from other proteins in wheat germ extract by DGU. This synthesis method is useful in the preparation of MP for structural and functional analysis.
Wheat Cell-free protein synthesis
Details of the wheat cell-free reaction were described in previously [28–30]. The 40 MPs and five soluble proteins in this study were selected from the Mammalian Gene Collection and FANTOM collection (Danaform, Yokohama, Japan). The unique primers for each protein (Additional file 1 Table S1) were designed and templates for transcription were made by the split-primer PCR method as described previously [18, 19]. The first PCR was performed with 10 nM of the gene specific primer (Additional file 1 Table S1) and 10 nM of the AODA2303 primer (5'-GTCAGACCCCGTAGAAAAGA) or 10 nM AODS primer (5'-TTTCTACGGGGTCTGACGCT). The second PCR was amplified with 100 nM SPu primer (5'-GCGTAGCATTTAGGTGACACT), 1 nM deSP6E01-S1 primer (5'-GGTGACACTATAGAACTCACCTATCTCCCCAACACCTAATAACATTCAAT CACTCTTTCCACTAACCACCTATCTACATCACCAACCACCCACCACCACCAATG), and 100 nM AODA2303 primer or 100 nM AODS primer. mRNA was prepared by in vitro transcription in a reaction volume of 100 μl and was purified by ethanol precipitation. The mRNA pellet was resuspended in 30 μl of water. The translation reaction was performed using the bilayer method, supplemented with [14C-leucine (50 μCi/ml, GE Healthcare, Tokyo, Japan) , in which a 25 μl translation layer was overlaid with a 125 μl substrate feeding buffer. Asolectin liposomes were prepared as described previously [16, 32] and added to both layers (10 mg/ml final concentration). The bilayer reaction was incubated at 26°C for 16 h. After the reaction, the amount of [14C]-leucine incorporation into synthesized proteins, an indicator of synthesis yield, was determined by 10% trichloroacetic acid precipitation and liquid scintillation spectroscopy.
Sucrose density gradient ultracentrifugation
Lipid/MP complexes were separated from proteins in the wheat germ extract by sucrose DGU. Synthesized proteins (100 μl) were loaded onto a discontinuous sucrose gradient consisting of 1,300 μl of 10% (w/v) and 600 μl of 30% (w/v) sucrose in DGU solution containing 140 mM NaCl, 5.4 mM KCl and 10 mM Tris-HCl (pH 8.0). The gradient was centrifuged at 105,000 g for 4 h at 4°C in a Hitachi S55S rotor (Hitachi High-Technology, Tokyo, Japan). Fractions were collected from the top of the tubes, and the protein content of each fraction was estimated by measurement of the amount of radioactivity associated with proteins precipitated by 10% trichloroacetic acid.
Transport assay of phosphate translocator
Liposomes were prepared from acetone-washed asolectin (Sigma-Aldrich, Tokyo, Japan) by sonication for 5 min at 4°C in water. AtPPT1 was synthesized by wheat cell-free system with (10 mg/ml final concentration) or without liposomes. Half of the AtPPT1 synthesized without liposomes was mixed with liposomes (10 mg/ml final concentration) after the synthesis reaction and incubated for 30 min at 26°C. These reaction mixtures were desalted by gel filtration with a MicroSpin G-25 column (GE Healthcare) that had been equilibrated with 10 mM Tricine-KOH (pH 7.6).
The amount of protein synthesized in the cell-free system was estimated from the incorporation of [14C]-leucine. After the reaction, the amount of [14C]-leucine incorporation into synthesized proteins, an indicator of synthesis yield, was determined by 10% trichloroacetic acid precipitation and liquid scintillation spectroscopy.
Substrate-including liposomes (80 mg/ml final concentration) were prepared from acetone-washed asolectin by sonication for 5 min at 4°C in a solution containing 200 mM Tricine-KOH (pH 7.6), 40 mM potassium gluconate and 60 mM potassium phosphate (substrate). Desalted reaction mixtures were mixed with the substrate-preloaded liposomes, frozen in liquid nitrogen, thawed at room temperature and sonicated for 18 sec. Substrate that remained outside of the liposomes was removed with a Dowex AG-1X8 column (Bio-Rad, Tokyo, Japan) that had been equilibrated with a solution containing 100 mM sodium gluconate, 40 mM potassium gluconate and 10 mM Tricine-KOH (pH 7.6). The liposome mixture was applied to the column and eluted with the equilibration solution.
Transport reactions were initiated by the addition of 13 μl of [32P] Pi (GE healthcare) to 300 μl of liposomes (final phosphate concentration, inside: 30 mM, outside: 0.5 mM). The assay was performed at 25°C for 2 min, and the reaction was terminated by application of the reaction mixture to a Dowex AG-1X8 column that had been equilibrated with 150 mM sodium acetate. The radio-activity associated with the eluted liposomes was measured with a liquid scintillation spectrometer.
GFP fragments were amplified by PCR with the primers EcoRV-GFP-5' (5'-GAGAGATATCATGGGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAG TGGCACGAAGGTGGAGGTGGAATGGTGAGCAAGGGCGAGGA-3') and GFP-NotI-3' (5'-TCTCGCGGCCGCTCCACCTCCACCCTTGTACAGCTCGTCCATGC-3). The PCR fragments were digested with EcoRV and NotI and then cloned into the corresponding sites in the pEU-E01-MCS vector (Cellfree Sciences, Matsuyama, Japan). The resultant plasmid was designated pEU-E01-GFP-N. P2RX4, P2RX1, and SLC6A18 were obtained from cDNA clones and amplified by PCR with the primer sets NotI-P2RX4-5' (5'-GAGAGCGGCCGCTGAAAACCTGTATTTTCAGGGCATGGCGGGCTGCTGC GCCGC) and P2RX4-SalI-3' (5'-AGAGGTCGACTCACTGGTCCAGCTCACTAG), NotI-P2RX1-5' (5'-GAGAGCGGCCGCTGAAAACCTGTATTTTCAGGGCATGGCGGGCTGCTGC GCCGC) and P2RX1-SalI-3' (5'-AGAGGTCGACTCACTGGTCCAGCTCACTAG), and NotI-SLC6A18-5' (5'-GAGAGCGGCCGCTGAAAACCTGTATTTTCAGGGCATGGCTCATGCCCCA GAACC) and SLC6A18-SalI-3' (5'-AGAGGTCGACTCAGCGCATGTCCGTGTCCG), respectively. These PCR fragments were digested with NotI and SalI and then cloned into the NotI-SalI sites of pEU-E01-GFP-N. The resultant plasmids were designated pUE-E01-GFP-P2RX4, pUE-E01-GFP-P2RX1, and pUE-E01-GFP-SLC6A18, respectively, and used for production of GFP-fusion proteins. The P2RX4 ORF was amplified by PCR using the primer pair P2RX4-EcoRV-5' (5'-GAGAGATATCATGGCGGGCTGCTGCGCCGC) and P2RX4-NotI-3' (5'-CTCTGCGGCCGCTCCACCTCCACCCTGGTCCAGCTCACTAGCAA). The PCR product was digested with EcoRV and NotI and then inserted into EcoRV-NotI sites of pEU-E01-MCS. The resultant plasmid was designated pEU-E01-P2RX4. The nucleotide sequences of each DNA fragment amplified by PCR was confirmed by DNA sequencing.
Detection and quantification of fluorescence from GFP proteins
Fluorescence images were visualized with a transilluminator (excitation: 400-500 nm, Dark Reader DR45M, Clare Chemical Research, Dolores, CO). Fluorescence intensity from GFP proteins was measured by a, Wallac 1420 Multilabel Counter spectrofluorometer (Perkin-Elmer Japan, Chiba, Japan).
Partial purification of P2RX4
mRNA was prepared by in vitro transcription using pEU-E01-P2RX4 as the template. The translation reaction was performed in the presence of liposomes (10 mg/ml final concentration) using the bilayer method with a bottom layer of 500 μl and an upper layer of 5500 μl. After the synthesis reaction, the reaction mixture was concentrated to 300 μl with a concentrator (Amicon Ultra-15, 30,000 MWCO, Millipore-Japan, Tokyo, Japan). For Accudenz (Accurate Chemical and Scientific, Westbury, NY) DGU, Accudenz was dissolved into DGU solution to make 30, 35, and 80% (w/v) Accudenz solutions. Three hundred μl of concentrated sample was mixed with 300 μl of 80% (w/v) Accudenz solution. The resultant 40% Accudenz solution containing the synthesized protein was placed in the bottom of a centrifuge tube, and overlaid with 650 μl of 35% (w/v) Accudenz solution, 650 μl of 30% (w/v) Accudenz solution, and 100 μl of DGU solution. The gradient was centrifuged at 105,000 g for 4 h at 4°C in a Hitachi S55S rotor (Hitachi High-Technology, Tokyo, Japan).
Coomassie Brilliant Blue
density gradient ultracentrifugation
human stearoyl-CoA desaturase 1
polymerase chain reaction
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
We greatly thank Naoki Nakagawa and Kohei Matsuda for technical assistances and Michael Andy Goren for proofreading this manuscript. This work was supported by the Special Coordination Funds for Promoting Science and Technology by the Ministry of Education, Culture, Sports, Science and Technology, Japan (YE and TS).
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