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Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis
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Journal of Biotechnology 133 (2008) 190–195
Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis Shin-ichiro M. Nomura a,b , Satoshi Kondoh a , Wakiko Asayama a,b , Akikazu Asada c , Shigemichi. Nishikawa c , Kazunari Akiyoshi a,b,∗ a
Institute of Biomaterials & Bioengineering, Tokyo Medical & Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan c WAKENYAKU CO., LTD., Research & Development Division, 945-1, Tsujide, Shimogasa-cho, Kusatsu-shi, Shiga 525-0029, Japan Received 26 January 2007; received in revised form 22 July 2007; accepted 3 August 2007
Abstract We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b5 (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b5 conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a “hydrophobic tag” for recruitment to the liposome surface. © 2007 Elsevier B.V. All rights reserved. Keywords: Giant liposome; Membrane protein; Cytochrome b5; In vitro protein synthesis; Wheat germ lysate
1. Introduction Membrane proteins play crucial roles in diverse biological functions such as signal transduction, energy production, and cellular communication. Understanding their structures and functions has been complicated by difficulties in isolating and purifying functional membrane proteins. In living systems, the folding and function of integrated membrane proteins are assisted by the microenvironment of a lipid bilayer of biomembranes. Therefore, liposomes have been frequently used as the basis for membrane protein reconstitution (Rigaud et al., 1995; Heginbotham et al., 1998; Furukawa and Haga, 2000; Salvail et al., 2002). We have been developing a novel method for directly obtaining pure membrane protein-containing liposomes (proteoliposomes). This entails cell-free protein synthesis was carried out in the presence of giant liposomes, i.e., an artificial cell∗ Corresponding author at: Institute of Biomaterials & Bioengineering, Tokyo Medical & Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: +81 3 5280 8020; fax: +81 3 5280 8027. E-mail address: [email protected] (K. Akiyoshi).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.023
model. Construction of an artificial cell-model is one of the useful approaches for understanding living cells (Yoshikawa and Nomura, 2000; Szostak et al., 2001; Noireaux et al., 2005; Luisi et al., 2006). Giant liposomes (phospholipid bilayer vesicles) with diameters of up to tens of micrometers have received considerable attention for the basis of such a cell-model because they can be visualized by optical microscopy (Hotani et al., 2003; Luisi and Lasic, 2000). Giant liposome encapsulated gene expression systems have recently been investigated (Tsumoto et al., 2001; Yu et al., 2001; Nomura et al., 2003; Noireaux and Libchaber, 2004; Ishikawa et al., 2004). In vitro gene expression that installed in a liposome can be regarded as a bootstrap sequence for a cell-model. In spite of the rapid advancement of in vitro gene expression systems, most of the research has been devoted to the synthesis of water soluble proteins (Shimizu et al., 2006; Vinarov et al., 2006; Nakano et al., 2004; Endo and Sawasaki, 2003). The synthesis of membrane proteins by a cell-free system is still limited (Klammt et al., 2006; Elbaz et al., 2004; Ishihara et al., 2005). Functional membrane protein expression with liposomes is a crucial step for constructing a more realistic cell-model and also for obtaining an understanding of the functions of membrane proteins.
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Fig. 1. Schematic illustrations of concept of the liposome displaying experiment. Synthesized nascent membrane proteins are expected to insert into the coexisting liposomal membranes.
In this study, the apo cytochrome b5 and its fusion proteins were selected as model membrane proteins. Cytochrome b5 is a ubiquitous electron transport hemoprotein, especially the microsomal and mitochondrial variants, which are membranebound proteins (Spatz and Strittmatter, 1971; Yubisui et al., 1988; Nakanishi et al., 2006). The protein has 133 amino acid residues with a polar domain and a hydrophobic membranebinding domain that comprises approximately 40 amino acid residues. It has been extensively used as a model membrane protein (Ladokhin et al., 1993). Apo cytochrome b5 (b5) and EGFP-conjugated b5 (EGFPb5) were synthesized by a wheat germ cell-free system in the presence of giant liposomes and were effectively displayed on the liposomal surface during the protein synthesis (Fig. 1). The proteo-liposome was isolated by simply centrifugation of the reaction mixture. We also report the presentation of water soluble enzymes to liposomes by conjugation of b5 as a “hydrophobic tag” for recruitment to the liposome surface. Cytochrome b5 conjugated dihydrofolate reductase (DHFR) was synthesized and displayed on the liposome surface. 2. Materials and methods 2.1. Materials Egg yolk phosphatidylcholine (EYPC) was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). 1,2-di-Oleoyl-sn-glycero3-phosphocholine (DOPC), 1,2-di-dimyristoyl-sn-glycero-3phosphocholine (DMPC), cholesterol were obtained from Sigma Co., Ltd. (St. Louis, MO). PROTEIOSTM , a wheat germ cellfree protein synthesis core kit, including the plasmids pEU3-NII, pEU3-DHFR, was purchased from WAKENYAKU Co., Ltd. (Shiga, Japan). 2.2. Liposome preparation and characterization Lipids were dissolved in the organic solvent (CHCl3 :CH3 OH = 2:1, v/v) to 100 mM. The lipids are EYPC, DOPC and DMPC/cholesterol = 7/3 in molar ratio. One milliliter of the lipid solution was set on a round bottomed flask,
and the solvent was evaporated. After vacuum drying (at least 1 h), a thin dried lipid film formed on the flask. The film was made to swell for liposome formation by adding 1 ml of 100 mM HEPES buffer solution for 30 min at 50 ◦ C. Cell-free expression buffer mix (PROTEIOS buffer #1:buffer #2:Nuclease free water, 26.75, 31.25, 67 in volume, respectively) 270 l and the liposome solution 30 l were applied to an ultrafiltration membrane (Microcon YM-3, Millipore), then centrifuged at 15,000 × g for 15 min. The ultrafiltration process was repeated three times to exchange the liposomal exterior solution with buffer mix. The final lipid concentration was diluted to 10 mM by the buffer mix. Size distribution of the obtained liposomes was investigated as follows. Liposomes encapsulating water soluble dye, calcein (1 mM in 100 mM HEPES buffer solution, as mentioned above) were prepared. One microliter of the liposome sample gently mixed with the 2.5 l of 50 mM Co(II)Cl2 solution for extra-liposomal quenching was sealed on the glass slide. The fluorescence images were obtained by the following setup: confocal laser-scanning unit (CSU10, Yokogawa) with an adapted inverted microscope (IX70, with lens objective UPlanSApo, 40×, Olympus) equipped with a cooled CCD camera (Hamamatsu). A sample was irradiated by diode laser (488 nm, 10 mW), then the signals were obtained through a band-pass filter (512–540 nm). The fluorescence images were randomly obtained for 20 focal image planes where at least three liposomes were present. The diameter of each liposome was estimated by particle-analysis software (Aquacosmos Ver.2.6, Hamamatsu). 2.3. Cell-free protein synthesis with liposomes A cell-free cytochrome b5 synthesis in the wheat germ cellfree kit was performed according to the instruction manual and developer’s report (Sawasaki et al., 2002) with slight modifications. Substrate mixture (250 l; PROTEIOS buffer #1 26.75 l, buffer #2 31.25 l, the 10 mM liposome solution 125 l and RNase free water (Promega) 67 l) and translation mixture (50 l; RNase free water 1.8 l, 10 mg ml−1 creatine kinase (Promega) 1.7 l, buffer #2 2.0 l, 40 U l−1 RNase inhibitor
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(Wako) 1.0 l, 1.4 g l−1 cytochrome b5 mRNA 8.5 l, wheat germ extract (PROTEIOS) 10 l, and the liposome solution 25 l), were prepared. Plasmid construction and mRNA preparation are described in the supporting information. The translation mixture was carefully set into the under layer of the substrate mixture in 96 wells of the titer plate and incubated at 26 ◦ C for 24 h. In the case of EGFP-conjugated cytochrome b5, the translation reaction time course was measured with a 96-well plate reader (Wallac 1420 ARVOsx, Perkin-Elmer. The filter set adopted was F485 and F535 for excitation and emission, respectively.) 2.4. Fluorescence microscopic observation An 8 l amount of the liposomal solution with the synthesized EGFP-b5 was sealed between the glass slide (24 × 60 mm, No. 1, Matsunami) and the cover slip (18 × 18, No.1, Matsunami) by nail-lacquer (Topcoat, Daiso). The sample was observed using an inverted fluorescent microscope (Olympus IX-70) equipped with a objective lens (Olympus 100 × ; UPlanApo NA. 1.35) and band-pass filter (U-MGFPHQ) for EGFP observation. The images were obtained by through a highly sensitive CCD camera system (C4742-95, HAMAMATSU). 2.5. Purification of the cytochrome b5 with liposome The giant liposomes were isolated from the reaction solution by simplified discontinuous density-gradient centrifugation as follows. A 50 l amount of the solution was carefully put on a 100 l of the 0.3 M sucrose solution. After centrifugation at 15,000 × g for 15 min at 4 ◦ C, precipitation was removed. 300 l of buffer mix was carefully put on 60 l of the supernatant. The sample was washed three times by centrifugation at 15,000 × g for 15 min at 4 ◦ C with buffer mix. After acetone treatment for removing lipids, SDS-PAGE was performed, with staining by bio-safe coomasie blue (Bio-rad). 2.6. Analysis of the synthesized DHFR-conjugated cytochrome b5 displayed on the liposome DHFR activity assay was performed according to Widemann et al., 1999. Assay buffer A was 0.5 mol l−1 Tris buffer, pH 7.5. The purified EYPC liposomal sample was desalted by ultrafiltration (Microcon YM-3, Millipore) 15,000 × g for 15 min by buffer A 200 l, three times. Stock solutions of dihydrofolate (FH2, 25 mg in 1.5 ml of 2-mercaptoethanol and 6.0 ml of buffer A), NADPH (50 mg in 10 ml of buffer A) were stored at −80 ◦ C. The NADPH/DHFR-b5-liposome reaction solution consisted of thawed 4 l aliquot NADPH stock solutions and the DHFR-b5 displayed-liposome solutions (30, 20, 4 l, mixed with buffer A to 0.5, 10.5, 26.5 l), respectively. Each reaction solution was adjusted to 34.5 l. The FH2 reaction solution (78 l) was added to 1 cm square cell. The sample was gently mixed by pipetting, after which the NADPH/DHFR-b5 -liposome reaction solution (30 l) was added. The absorbance of each sample was read in the UV/vis spectrometer V-560 (JASCO) at room temperature
at wavelengths of 340 nm with a reading interval of 20 s for a duration of 20 min. The relative enzyme activity was calculated from the slopes of obtained time cause data. 3. Results and discussion Cell-free synthesis of EGFP-conjugated apo cytochrome b5 (EGFP-b5) was carried out with the wheat germ cell-free system in the presence of liposomes. The time course of the reaction was followed by EGFP fluorescence (Fig. 2). The final yields of proteins slightly increased in the presence of liposomes, though the rate of the reaction did not change much. Fig. 3 shows the fluorescence microscopic images of the reaction mixture of EGFP-b5 synthesis in the presence of giant liposomes. The image analysis supports the expressed EGFP-b5 proteins being attached primarily in the giant liposomal membranes. The size distribution of the liposomes used in this study was evaluated by the fluorescence quenching method. Fig. 3b shows the size distribution of the liposomes loaded into the reaction mixture. The average mean diameters are as follows: EYPC, 4.2 m; DOPC, 3.4 m. The estimated mode values from the histograms are 2 m for each. From the fluorescence microscopic images, significant differences in the liposome sizes and shapes were not observed under protein synthesis. The giant liposomes can be easily isolated from the reaction mixture by simplified discontinuous density-gradient centrifugation, although the small-sized liposomes must be separated using gel-filtration. After centrifugation of the reaction mixture of EGFP-b5 in the presence of the giant liposomes, the pellets of liposome fractions were analyzed by SDS page (Fig. 4, comasie blue staining). In the liposomal fractions (Fig. 4b, lane 3 DOPC liposome, lane 4 eggPC), the b5 band was mainly observed. By the simplified density-gradient centrifugation method, the membrane protein, b5 could be easily purified from the reaction mixtures in the case of both DOPC and egg yolk PC liposomes. Similar results were obtained in the case of apo cytchrome b5 , which is not conjugated by EGFP (Fig. 4a, lane 3 DOPC liposome, lane 4 eggPC). There are few reports that membrane proteins are directly reconstituted into the liposomal membrane by cell-free synthesis. Noireaux and Libchaber reported that a functional membrane protein of bacterial ␣-hemolysin was synthesized in the presence of giant liposomes by E. coli extract (Noireaux and Libchaber, 2004). Recently, we succeed in expressing and displaying mam-
Fig. 2. Time course of the fluorescence intensity of the EGFP-conjugated apo cytochrome b5 in vitro translation reaction in the presence of liposomes.
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Fig. 3. (a) Fluorescence microscopic images of EGFP-b5 displaying giant liposomes. Left, EYPC; right, DOPC liposome. The high initial lipid concentration tends to make the polymorphic liposomes. Scale bar = 5 m. (b) Size distribution of the liposomes loaded to the protein synthesis reaction solution. The frequency was normalized.
malian membrane protein connexin 43 into giant liposomes (unpublished result). In living systems, most mammalian membrane proteins are synthesized and folded at the microsomal membrane, assisting by a special complex called the “translocon” (Alder and Johnson, 2004). On the other hand, interaction between the bilayer membrane and membrane proteins has been widely discussed in the fields of molecular assembly (Owicki et al., 1978; White and von Heijne, 2005) and biosensor applications (Anrather et al., 2004; Cornell et al., 1997). Since membrane proteins are regarded as amphiphilic polymers, in vitro membrane proteins should directly interact with liposomes by hydrophobic interaction from the thermodynamic point of view. During the expression of b5 by the protein synthesis
machinery, hydrophobic b5 may spontaneously interact with liposomes by hydrophobic interaction. Hanlon et al. (2000) reported that the membrane domain of b5 interacted to liposomes as trans-membrane type, not as hair-pinned membrane anchor type. The orientation and conformation of synthesized membrane proteins in the liposome are under investigation in our laboratory. Presenting functional proteins on the membrane surface is of growing interest in nanobiotechnology (Kodadek, 2001; Predki, 2004; Fang et al., 2002). By using apo cytochrome b5 as a hydrophobic tag, conjugated water soluble enzymes can be presented on the liposomal surface. To this end, cytochrome b5 conjugated dihydrofolate reductase was synthesized by the same
Fig. 4. SDS-PAGE analysis of in vitro translation of cytochrome b5 with liposomes. The gels were stained by coomassie brilliant blue. (a) Synthesized apo cytochrome b5. Lanes 1 and 3, before and after purification by simplified density-gradient centrifugation with DOPC liposomes. Lanes 2 and 4, with egg yolk PC liposomes. (b) Synthesized EGFP-conjugated apo cytochrome b5. Lanes 1 and 3, before and after purification with DOPC liposomes. Lanes 2 and 4, with egg yolk PC liposomes.
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Fig. 5. DHFR-conjugated cytochromes b5 displayed on EYPC liposomes. (a) SDS-PAGE analysis. Lanes 1 and 3, before purification. Lanes 2 and 4, after the purification procedure without and with mRNA of b5. (b) Schematic illustration of the analysis of enzymatic activity of DHFR-conjugated b5 on liposomes. (c) Enzymatic activity of synthesized DHFR-conjugated apo cytochrome b5 on liposomes. The values of relative enzyme activity were obtained from the 340 nm absorbance slopes of their time courses.
procedure. The pure proteo-liposome (made from EYPC) containing DHFR-conjugated b5 was isolated from the reaction solution by simplified discontinuous density-gradient centrifugation (Fig. 5a, lane 4). Synthesized b5 acts as a “hydrophobic tag” for recruitment to the liposome surface. Fig. 5b shows the schematic illustration for analyzing the DHFR enzyme activity assay. The assay is based on the conversion of dihydrofolate (FH2 ) to tetrahydrofolate and the resulting oxidation of NADPH to NADP+ , which was monitored by measurement of the absorbance at 340 nm (Widemann et al., 1999). Fig. 5c shows the enzymatic activities of DHFR-conjugated cytochrome b5 at various concentrations of the reaction samples. This suggests that conjugated-DHFR is located in the native form on the liposome surface. In conclusion, we present here a novel approach for salvaging nascent membrane proteins by cell-free synthesis with giant liposomes. The method also uses a hydrophobic tag for immobilization of the specific hydrophilic enzyme on the liposomal surface. These methods are useful for understanding the functions of membrane proteins and also for constructing realistic cell-models. Acknowledgements This research was supported by a grant from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and Dental University. This work was also supported by Grants-in-Aid for Scientific Research from the Japanese Government (Nos. 18GS0421, 18048013, 18048014). Pacific Edit reviewed the manuscript prior to submission. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiotec.2007.08.023. References Alder, N.N., Johnson, A.E., 2004. Cotranslational membrane protein biogenesis at the endoplasmic reticulum. J. Biol. Chem. 279, 22787–22790.
Anrather, D., Smetazko, M., Saba, M., Alguel, Y., Schalkhammer, T., 2004. Supported membrane nanodevices. J. Nanosci. Nanotech. 4 (1–2), 1–22. Cornell, B.A., BraachMaksvytis, V.L.B., King, L.G., Osman, P.D.J., Raguse, B., Wieczorek, L., Pace, R.J., 1997. A biosensor that uses ion-channel switches. Nature 387 (6633), 580–583. Elbaz, Y., Steiner-Mordoch, S., Danieli, T., Schuldiner, S., 2004. In vitro synthesis of fully functional EmrE, a multidrug transporter, and study of its oligomeric state. Proc. Natl. Acad. Sci. U.S.A. 101 (6), 1519–1524. Endo, Y., Sawasaki, T., 2003. High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system. Biotechnol. Adv. 21 (8), 695–713. Fang, Y., Frutos, A.G., Lahiri, J., 2002. Membrane protein microarrays. J. Am. Chem. Soc. 124, 2394–2395. Furukawa, H., Haga, T., 2000. Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli. J. Biochem. 127, 151–161. Hanlon, M.R., Begum, R.R., Newbold, R.J., Whitford, D., Wallace, B.A., 2000. In vitro membrane-inserted conformation of the cytochrome b5 tail. Biochem. J. 352, 117–124. Heginbotham, L., Kolmakova-Partensky, L., Miller, C., 1998. Functional reconstitution of a prokaryotic K+ channel. J. Gen. Physiol. 111, 741–749. Hotani, H., Inaba, T., Nomura, F., Takeda, S., Takiguchi, K., Itoh, T.J., Umeda, T., Ishijima, A., 2003. Mechanical analyses of morphological and topological transformation of liposomes. BioSystems 71, 93–100. Ishihara, G., Goto, M., Saeki, M., Ito, K., Hori, T., Kigawa, T., Shirouzu, M., Yokoyama, S., 2005. Expression of G protein coupled receptors in a cellfree translational system using detergents and thioredoxin-fusion vectors. Protein Expr. Purif. 41 (1), 27–37. Ishikawa, K., Sato, K., Shima, Y., Urabe, I., Yomo, T., 2004. Expression of a cascading genetic network within liposomes. FEBS Lett. 576, 387–390. Klammt, C., Schwarz, D., Lohr, F., Schneider, B., Dotsch, V., Bernhard, F., 2006. Cell-free expression as an emerging technique for the large scale production of integral membrane protein. FEBS J. 273 (18), 4141–4153. Kodadek, T., 2001. Protein microarrays: prospects and problems. Chem. Biol. 8 (2), 105–115. Ladokhin, A.S., Wang, L., Steggles, A.W., Malak, H., Holloway, P.W., 1993. Fluorescence study of a temperature-induced conversion from the “Loose” to the “Tight” binding form of membrane-bound Cytochrome b5. Biochemistry 32, 6951–6956. Luisi, P.L., Ferri, F., Stano, P., 2006. Approaches to semi-synthetic minimal cells: a review. Naturwissenschaften 93 (1), 1–13. Luisi, P.L., Lasic, D.D., 2000. In: Luisi, P.L., Walde, P. (Eds.), Giant Vesicles, Perspectives in Supramolecular Chemistry. Wiley, Chichester, pp. 3–24. Nakanishi, N., Takeuchi, F., Okamoto, H., Tamura, A., Hori, H., Tsubaki, M., 2006. Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings. J. Biochem. (Tokyo) 140, 561–571. Nakano, H., Kawarasaki, Y., Yamane, T., 2004. Cell-free protein synthesis systems: increasing their performance and applications. Adv. Biochem. Eng. Biotechnol. 90, 135–149.
S.-i.M. Nomura et al. / Journal of Biotechnology 133 (2008) 190–195 Noireaux, V., Libchaber, A., 2004. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. U.S.A. 101 (51), 17669–17674. Noireaux, V., Bar-Ziv, R., Godefroy, J., Salman, H., Libchaber, A., 2005. Toward an artificial cell based on gene expression in vesicles. Phys. Biol. 2 (3), 1–8. Nomura, S.-i.M., Tsumoto, K., Hamada, T., Akiyoshi, K., Nakatani, Y., Yoshikawa, K., 2003. Gene expression within cell-sized lipid vesicles. ChemBioChem 4, 1172–1175. Owicki, J.C., Springgate, M.W., McConnell, H.M., 1978. Theoretical study of protein–lipid interactions in bilayer membranes. Proc. Natl. Acad. Sci. U.S.A. 75, 1616–1619. Predki, P.F., 2004. Functional protein microarrays: ripe for discovery. Curr. Opin. Chem. Biol. 8, 8–13. Rigaud, J.-L., Pitard, B., Levy, D., 1995. Reconstitution of membrane proteins into liposomes: application to energy-transducing membrane proteins. Biochim. Biophys. Acta Bioenerg. 1231, 223–246. Salvail, D., Cloutier, M., Rousseau, E., 2002. Functional reconstitution of an eicosanoid-modulated Cl-channel from bovine tracheal smooth muscle. Am. J. Physiol. Cell Physiol. 282, C567–C577. Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kamura, N., Ogasawara, T., Kuroita, T., Endo, Y., 2002. A bilayer cell-free protein synthesis system for high-throughput screening of gene products. FEBS Lett. 514, 102–105. Shimizu, Y., Kuruma, Y., Ying, B.-W., Umekage, S., Ueda, T., 2006. Cell-free translation systems for protein engineering. FEBS J. 273, 4133–4140.
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Journal of Biotechnology 133 (2008) 190–195
Direct preparation of giant proteo-liposomes by in vitro membrane protein synthesis Shin-ichiro M. Nomura a,b , Satoshi Kondoh a , Wakiko Asayama a,b , Akikazu Asada c , Shigemichi. Nishikawa c , Kazunari Akiyoshi a,b,∗ a
Institute of Biomaterials & Bioengineering, Tokyo Medical & Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan b Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, Tokyo, Japan c WAKENYAKU CO., LTD., Research & Development Division, 945-1, Tsujide, Shimogasa-cho, Kusatsu-shi, Shiga 525-0029, Japan Received 26 January 2007; received in revised form 22 July 2007; accepted 3 August 2007
Abstract We investigated the direct constitution of membrane proteins into giant liposomes in cell-free (in vitro) protein synthesis. Giant liposomes were present in a translation reaction cocktail of a wheat germ cell-free protein translation system. Apo cytochrome b5 (b5) and its fusion proteins were synthesized and directly localized in the liposomes. After the translation reaction, the proteo-liposomes were isolated by simplified discontinuous density-gradient centrifugation. Apo cytochrome b5 conjugated dihydrofolate reductase (DHFR) was synthesized in the same procedure and the protein was directly displayed on the liposome surface. b5 acts as a “hydrophobic tag” for recruitment to the liposome surface. © 2007 Elsevier B.V. All rights reserved. Keywords: Giant liposome; Membrane protein; Cytochrome b5; In vitro protein synthesis; Wheat germ lysate
1. Introduction Membrane proteins play crucial roles in diverse biological functions such as signal transduction, energy production, and cellular communication. Understanding their structures and functions has been complicated by difficulties in isolating and purifying functional membrane proteins. In living systems, the folding and function of integrated membrane proteins are assisted by the microenvironment of a lipid bilayer of biomembranes. Therefore, liposomes have been frequently used as the basis for membrane protein reconstitution (Rigaud et al., 1995; Heginbotham et al., 1998; Furukawa and Haga, 2000; Salvail et al., 2002). We have been developing a novel method for directly obtaining pure membrane protein-containing liposomes (proteoliposomes). This entails cell-free protein synthesis was carried out in the presence of giant liposomes, i.e., an artificial cell∗ Corresponding author at: Institute of Biomaterials & Bioengineering, Tokyo Medical & Dental University, 2-3-10, Kanda-surugadai, Chiyoda-ku, Tokyo 101-0062, Japan. Tel.: +81 3 5280 8020; fax: +81 3 5280 8027. E-mail address: [email protected] (K. Akiyoshi).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.023
model. Construction of an artificial cell-model is one of the useful approaches for understanding living cells (Yoshikawa and Nomura, 2000; Szostak et al., 2001; Noireaux et al., 2005; Luisi et al., 2006). Giant liposomes (phospholipid bilayer vesicles) with diameters of up to tens of micrometers have received considerable attention for the basis of such a cell-model because they can be visualized by optical microscopy (Hotani et al., 2003; Luisi and Lasic, 2000). Giant liposome encapsulated gene expression systems have recently been investigated (Tsumoto et al., 2001; Yu et al., 2001; Nomura et al., 2003; Noireaux and Libchaber, 2004; Ishikawa et al., 2004). In vitro gene expression that installed in a liposome can be regarded as a bootstrap sequence for a cell-model. In spite of the rapid advancement of in vitro gene expression systems, most of the research has been devoted to the synthesis of water soluble proteins (Shimizu et al., 2006; Vinarov et al., 2006; Nakano et al., 2004; Endo and Sawasaki, 2003). The synthesis of membrane proteins by a cell-free system is still limited (Klammt et al., 2006; Elbaz et al., 2004; Ishihara et al., 2005). Functional membrane protein expression with liposomes is a crucial step for constructing a more realistic cell-model and also for obtaining an understanding of the functions of membrane proteins.
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Fig. 1. Schematic illustrations of concept of the liposome displaying experiment. Synthesized nascent membrane proteins are expected to insert into the coexisting liposomal membranes.
In this study, the apo cytochrome b5 and its fusion proteins were selected as model membrane proteins. Cytochrome b5 is a ubiquitous electron transport hemoprotein, especially the microsomal and mitochondrial variants, which are membranebound proteins (Spatz and Strittmatter, 1971; Yubisui et al., 1988; Nakanishi et al., 2006). The protein has 133 amino acid residues with a polar domain and a hydrophobic membranebinding domain that comprises approximately 40 amino acid residues. It has been extensively used as a model membrane protein (Ladokhin et al., 1993). Apo cytochrome b5 (b5) and EGFP-conjugated b5 (EGFPb5) were synthesized by a wheat germ cell-free system in the presence of giant liposomes and were effectively displayed on the liposomal surface during the protein synthesis (Fig. 1). The proteo-liposome was isolated by simply centrifugation of the reaction mixture. We also report the presentation of water soluble enzymes to liposomes by conjugation of b5 as a “hydrophobic tag” for recruitment to the liposome surface. Cytochrome b5 conjugated dihydrofolate reductase (DHFR) was synthesized and displayed on the liposome surface. 2. Materials and methods 2.1. Materials Egg yolk phosphatidylcholine (EYPC) was obtained from Funakoshi Co., Ltd. (Tokyo, Japan). 1,2-di-Oleoyl-sn-glycero3-phosphocholine (DOPC), 1,2-di-dimyristoyl-sn-glycero-3phosphocholine (DMPC), cholesterol were obtained from Sigma Co., Ltd. (St. Louis, MO). PROTEIOSTM , a wheat germ cellfree protein synthesis core kit, including the plasmids pEU3-NII, pEU3-DHFR, was purchased from WAKENYAKU Co., Ltd. (Shiga, Japan). 2.2. Liposome preparation and characterization Lipids were dissolved in the organic solvent (CHCl3 :CH3 OH = 2:1, v/v) to 100 mM. The lipids are EYPC, DOPC and DMPC/cholesterol = 7/3 in molar ratio. One milliliter of the lipid solution was set on a round bottomed flask,
and the solvent was evaporated. After vacuum drying (at least 1 h), a thin dried lipid film formed on the flask. The film was made to swell for liposome formation by adding 1 ml of 100 mM HEPES buffer solution for 30 min at 50 ◦ C. Cell-free expression buffer mix (PROTEIOS buffer #1:buffer #2:Nuclease free water, 26.75, 31.25, 67 in volume, respectively) 270 l and the liposome solution 30 l were applied to an ultrafiltration membrane (Microcon YM-3, Millipore), then centrifuged at 15,000 × g for 15 min. The ultrafiltration process was repeated three times to exchange the liposomal exterior solution with buffer mix. The final lipid concentration was diluted to 10 mM by the buffer mix. Size distribution of the obtained liposomes was investigated as follows. Liposomes encapsulating water soluble dye, calcein (1 mM in 100 mM HEPES buffer solution, as mentioned above) were prepared. One microliter of the liposome sample gently mixed with the 2.5 l of 50 mM Co(II)Cl2 solution for extra-liposomal quenching was sealed on the glass slide. The fluorescence images were obtained by the following setup: confocal laser-scanning unit (CSU10, Yokogawa) with an adapted inverted microscope (IX70, with lens objective UPlanSApo, 40×, Olympus) equipped with a cooled CCD camera (Hamamatsu). A sample was irradiated by diode laser (488 nm, 10 mW), then the signals were obtained through a band-pass filter (512–540 nm). The fluorescence images were randomly obtained for 20 focal image planes where at least three liposomes were present. The diameter of each liposome was estimated by particle-analysis software (Aquacosmos Ver.2.6, Hamamatsu). 2.3. Cell-free protein synthesis with liposomes A cell-free cytochrome b5 synthesis in the wheat germ cellfree kit was performed according to the instruction manual and developer’s report (Sawasaki et al., 2002) with slight modifications. Substrate mixture (250 l; PROTEIOS buffer #1 26.75 l, buffer #2 31.25 l, the 10 mM liposome solution 125 l and RNase free water (Promega) 67 l) and translation mixture (50 l; RNase free water 1.8 l, 10 mg ml−1 creatine kinase (Promega) 1.7 l, buffer #2 2.0 l, 40 U l−1 RNase inhibitor
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(Wako) 1.0 l, 1.4 g l−1 cytochrome b5 mRNA 8.5 l, wheat germ extract (PROTEIOS) 10 l, and the liposome solution 25 l), were prepared. Plasmid construction and mRNA preparation are described in the supporting information. The translation mixture was carefully set into the under layer of the substrate mixture in 96 wells of the titer plate and incubated at 26 ◦ C for 24 h. In the case of EGFP-conjugated cytochrome b5, the translation reaction time course was measured with a 96-well plate reader (Wallac 1420 ARVOsx, Perkin-Elmer. The filter set adopted was F485 and F535 for excitation and emission, respectively.) 2.4. Fluorescence microscopic observation An 8 l amount of the liposomal solution with the synthesized EGFP-b5 was sealed between the glass slide (24 × 60 mm, No. 1, Matsunami) and the cover slip (18 × 18, No.1, Matsunami) by nail-lacquer (Topcoat, Daiso). The sample was observed using an inverted fluorescent microscope (Olympus IX-70) equipped with a objective lens (Olympus 100 × ; UPlanApo NA. 1.35) and band-pass filter (U-MGFPHQ) for EGFP observation. The images were obtained by through a highly sensitive CCD camera system (C4742-95, HAMAMATSU). 2.5. Purification of the cytochrome b5 with liposome The giant liposomes were isolated from the reaction solution by simplified discontinuous density-gradient centrifugation as follows. A 50 l amount of the solution was carefully put on a 100 l of the 0.3 M sucrose solution. After centrifugation at 15,000 × g for 15 min at 4 ◦ C, precipitation was removed. 300 l of buffer mix was carefully put on 60 l of the supernatant. The sample was washed three times by centrifugation at 15,000 × g for 15 min at 4 ◦ C with buffer mix. After acetone treatment for removing lipids, SDS-PAGE was performed, with staining by bio-safe coomasie blue (Bio-rad). 2.6. Analysis of the synthesized DHFR-conjugated cytochrome b5 displayed on the liposome DHFR activity assay was performed according to Widemann et al., 1999. Assay buffer A was 0.5 mol l−1 Tris buffer, pH 7.5. The purified EYPC liposomal sample was desalted by ultrafiltration (Microcon YM-3, Millipore) 15,000 × g for 15 min by buffer A 200 l, three times. Stock solutions of dihydrofolate (FH2, 25 mg in 1.5 ml of 2-mercaptoethanol and 6.0 ml of buffer A), NADPH (50 mg in 10 ml of buffer A) were stored at −80 ◦ C. The NADPH/DHFR-b5-liposome reaction solution consisted of thawed 4 l aliquot NADPH stock solutions and the DHFR-b5 displayed-liposome solutions (30, 20, 4 l, mixed with buffer A to 0.5, 10.5, 26.5 l), respectively. Each reaction solution was adjusted to 34.5 l. The FH2 reaction solution (78 l) was added to 1 cm square cell. The sample was gently mixed by pipetting, after which the NADPH/DHFR-b5 -liposome reaction solution (30 l) was added. The absorbance of each sample was read in the UV/vis spectrometer V-560 (JASCO) at room temperature
at wavelengths of 340 nm with a reading interval of 20 s for a duration of 20 min. The relative enzyme activity was calculated from the slopes of obtained time cause data. 3. Results and discussion Cell-free synthesis of EGFP-conjugated apo cytochrome b5 (EGFP-b5) was carried out with the wheat germ cell-free system in the presence of liposomes. The time course of the reaction was followed by EGFP fluorescence (Fig. 2). The final yields of proteins slightly increased in the presence of liposomes, though the rate of the reaction did not change much. Fig. 3 shows the fluorescence microscopic images of the reaction mixture of EGFP-b5 synthesis in the presence of giant liposomes. The image analysis supports the expressed EGFP-b5 proteins being attached primarily in the giant liposomal membranes. The size distribution of the liposomes used in this study was evaluated by the fluorescence quenching method. Fig. 3b shows the size distribution of the liposomes loaded into the reaction mixture. The average mean diameters are as follows: EYPC, 4.2 m; DOPC, 3.4 m. The estimated mode values from the histograms are 2 m for each. From the fluorescence microscopic images, significant differences in the liposome sizes and shapes were not observed under protein synthesis. The giant liposomes can be easily isolated from the reaction mixture by simplified discontinuous density-gradient centrifugation, although the small-sized liposomes must be separated using gel-filtration. After centrifugation of the reaction mixture of EGFP-b5 in the presence of the giant liposomes, the pellets of liposome fractions were analyzed by SDS page (Fig. 4, comasie blue staining). In the liposomal fractions (Fig. 4b, lane 3 DOPC liposome, lane 4 eggPC), the b5 band was mainly observed. By the simplified density-gradient centrifugation method, the membrane protein, b5 could be easily purified from the reaction mixtures in the case of both DOPC and egg yolk PC liposomes. Similar results were obtained in the case of apo cytchrome b5 , which is not conjugated by EGFP (Fig. 4a, lane 3 DOPC liposome, lane 4 eggPC). There are few reports that membrane proteins are directly reconstituted into the liposomal membrane by cell-free synthesis. Noireaux and Libchaber reported that a functional membrane protein of bacterial ␣-hemolysin was synthesized in the presence of giant liposomes by E. coli extract (Noireaux and Libchaber, 2004). Recently, we succeed in expressing and displaying mam-
Fig. 2. Time course of the fluorescence intensity of the EGFP-conjugated apo cytochrome b5 in vitro translation reaction in the presence of liposomes.
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Fig. 3. (a) Fluorescence microscopic images of EGFP-b5 displaying giant liposomes. Left, EYPC; right, DOPC liposome. The high initial lipid concentration tends to make the polymorphic liposomes. Scale bar = 5 m. (b) Size distribution of the liposomes loaded to the protein synthesis reaction solution. The frequency was normalized.
malian membrane protein connexin 43 into giant liposomes (unpublished result). In living systems, most mammalian membrane proteins are synthesized and folded at the microsomal membrane, assisting by a special complex called the “translocon” (Alder and Johnson, 2004). On the other hand, interaction between the bilayer membrane and membrane proteins has been widely discussed in the fields of molecular assembly (Owicki et al., 1978; White and von Heijne, 2005) and biosensor applications (Anrather et al., 2004; Cornell et al., 1997). Since membrane proteins are regarded as amphiphilic polymers, in vitro membrane proteins should directly interact with liposomes by hydrophobic interaction from the thermodynamic point of view. During the expression of b5 by the protein synthesis
machinery, hydrophobic b5 may spontaneously interact with liposomes by hydrophobic interaction. Hanlon et al. (2000) reported that the membrane domain of b5 interacted to liposomes as trans-membrane type, not as hair-pinned membrane anchor type. The orientation and conformation of synthesized membrane proteins in the liposome are under investigation in our laboratory. Presenting functional proteins on the membrane surface is of growing interest in nanobiotechnology (Kodadek, 2001; Predki, 2004; Fang et al., 2002). By using apo cytochrome b5 as a hydrophobic tag, conjugated water soluble enzymes can be presented on the liposomal surface. To this end, cytochrome b5 conjugated dihydrofolate reductase was synthesized by the same
Fig. 4. SDS-PAGE analysis of in vitro translation of cytochrome b5 with liposomes. The gels were stained by coomassie brilliant blue. (a) Synthesized apo cytochrome b5. Lanes 1 and 3, before and after purification by simplified density-gradient centrifugation with DOPC liposomes. Lanes 2 and 4, with egg yolk PC liposomes. (b) Synthesized EGFP-conjugated apo cytochrome b5. Lanes 1 and 3, before and after purification with DOPC liposomes. Lanes 2 and 4, with egg yolk PC liposomes.
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Fig. 5. DHFR-conjugated cytochromes b5 displayed on EYPC liposomes. (a) SDS-PAGE analysis. Lanes 1 and 3, before purification. Lanes 2 and 4, after the purification procedure without and with mRNA of b5. (b) Schematic illustration of the analysis of enzymatic activity of DHFR-conjugated b5 on liposomes. (c) Enzymatic activity of synthesized DHFR-conjugated apo cytochrome b5 on liposomes. The values of relative enzyme activity were obtained from the 340 nm absorbance slopes of their time courses.
procedure. The pure proteo-liposome (made from EYPC) containing DHFR-conjugated b5 was isolated from the reaction solution by simplified discontinuous density-gradient centrifugation (Fig. 5a, lane 4). Synthesized b5 acts as a “hydrophobic tag” for recruitment to the liposome surface. Fig. 5b shows the schematic illustration for analyzing the DHFR enzyme activity assay. The assay is based on the conversion of dihydrofolate (FH2 ) to tetrahydrofolate and the resulting oxidation of NADPH to NADP+ , which was monitored by measurement of the absorbance at 340 nm (Widemann et al., 1999). Fig. 5c shows the enzymatic activities of DHFR-conjugated cytochrome b5 at various concentrations of the reaction samples. This suggests that conjugated-DHFR is located in the native form on the liposome surface. In conclusion, we present here a novel approach for salvaging nascent membrane proteins by cell-free synthesis with giant liposomes. The method also uses a hydrophobic tag for immobilization of the specific hydrophilic enzyme on the liposomal surface. These methods are useful for understanding the functions of membrane proteins and also for constructing realistic cell-models. Acknowledgements This research was supported by a grant from the Center of Excellence Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and Dental University. This work was also supported by Grants-in-Aid for Scientific Research from the Japanese Government (Nos. 18GS0421, 18048013, 18048014). Pacific Edit reviewed the manuscript prior to submission. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jbiotec.2007.08.023. References Alder, N.N., Johnson, A.E., 2004. Cotranslational membrane protein biogenesis at the endoplasmic reticulum. J. Biol. Chem. 279, 22787–22790.
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