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Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e4, 2014 www.elsevier.com/locate/jbiosc
Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system Yasuaki Kazuta,1 Tomoaki Matsuura,1, 2 Norikazu Ichihashi,1 and Tetsuya Yomo1, 3, 4, * Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan,2 Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan,3 and Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan4 Received 20 February 2014; accepted 23 April 2014 Available online xxx
In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and b-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro protein synthesis; PURE system; Green fluorescent protein; Dialysis; Synthetic biology]
Cell-free protein synthesis enables in vitro protein synthesis without the requirement of living cells (1e3). Because the cell-free system is independent of cell growth, the synthesis of proteins that may affect cell growth is possible. Furthermore, rapid protein production is possible using a cell-free system, as protein synthesis typically requires only a few hours. Because of these favorable properties, cell-free systems have been used for various applications and basic research. Among cell-free systems, the PURE system is the only fully reconstituted system in which the minimal components for protein translation are individually purified and reconstituted in vitro (4,5). Using this reconstituted system, components can be added or omitted, and the concentration of each component can be altered. This system has enabled the efficient incorporation of unnatural amino acids into peptides (6,7). Because of the inherently low nuclease and protease activity, this system has been used for the in vitro evolution of proteins and enzymes (8e10). An additional advantage of the PURE system is the lack of amino acid scrambling, which enables nuclear magnetic resonance structural studies (11,12). However, despite its wide application, there have been few reports on the enhancement of the yield of proteins synthesized using this reconstituted in vitro protein synthesis system (13,14). In this study, we report the optimization of the PURE system, which resulted in the synthesis of greater than 4 mg/mL protein. This optimization was achieved by modifying the component
* Corresponding author at: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: þ81 6 6879 4171; fax: þ81 6 6879 7433. E-mail address: [email protected] (T. Yomo).
concentrations and utilizing the system in dialysis mode. We have improved the protein yield of this system by including additional components and altering the component concentrations (15,16). Attempts to improve the yield of in vitro protein synthesis have been described (13,14). However, the development of a reconstituted in vitro protein synthesis system capable of synthesizing a protein at concentrations of greater than 1 mg/mL has not yet been achieved. We evaluated the performance of the optimized system using three different sets of component concentrations determined from the optimization process and demonstrate that milligram quantities of protein can be synthesized. MATERIALS AND METHODS DNA and RNA preparation The plasmid DNA used for the in vitro transcription-translation system was purified using the Qiagen Plasmid Mini Kit (Qiagen) according to the manufacturer’s instructions. Under the control of a T7 promoter, pETG5tag encoding green fluorescent protein (GFP), pET-dhfr (Gene Frontier) encoding dihydrofolate reductase (DHFR), and pET-bgal encoding bgalactosidase were used as expression plasmids. For in vitro transcription, pETG5tag was amplified using the primers T7F (50 -TAATACGACTCACTATAGGG-30 ) and T7R (50 GCTAGTTATTGCTCAGCGG-30 ) and KOD FX DNA polymerase (Toyobo, Osaka, Japan). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. RNA was prepared from this PCR product using the MEGAscript T7 Kit (Life Technologies) and purified using an RNeasy kit (Qiagen). Component preparation Versions 1 and 2 of the system and their components were prepared as previously described (5,15). The protein components constituting version 7 of the system were prepared with additional treatment. Components, with the exception of the ribosome, were stored in stock buffer (50 mM HEPES-KOH, pH 7.6, 100 mM KCl, 10 mM MgCl2, 30% glycerol, and 7 mM 2-mercaptoethanol), and the ribosome was stored in 70S buffer (20 mM HEPESKOH, pH 7.6, 6 mM Mg(OAc)2, 30 mM KCl, and 7 mM 2-mercaptoethanol). All of these components were dialyzed against buffer A (50 mM HEPES, pH 7.6, 10 mM
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
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J. BIOSCI. BIOENG., obtain a standard curve of the relationship between the volume of 35S-Met and the signal intensity obtained in the autoradiogram. Subsequently, an appropriate volume of 35S-Met (e.g., p ml/mL) was added to the reaction mixture in which a large excess of cold Met (0.3 mM) was present. After the synthesis reaction, a portion of the sample was subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. The volume of 35S-Met (e.g., q ml/mL) was determined from the band intensity in the autoradiogram using the standard curve. The concentration of incorporated Met was calculated using the equation 0.3 (q/p) (mM), and we obtained the concentration of the synthesized proteins by dividing this value by the number of Met residues in the sequence of each synthesized protein.
Fluorescent GFP concentration ( M)
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PURE ver 1 PURE ver 2 PURE ver 7
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RESULTS AND DISCUSSION
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FIG. 1. GFP synthesis using various versions of the reconstituted in vitro protein synthesis system. Time course of GFP synthesis in batch mode. RNA was used as a template at a concentration of 200 nM. GFP concentrations were determined from fluorescence intensities using the relationship between the fluorescence and concentration of GFP purified from E. coli. Purified GFP was assumed to be 100% active.
Mg(OAc)2, 100 mM potassium glutamate (K-Glu), and 7 mM 2-mercaptoethanol) using a microscale dialyzer (Nippon Genetics) with a molecular weight cut-off of 3000 Da at 4 C overnight. When the components precipitated as a result of dialysis, 3 M K-Glu was added until the pellets were dissolved. All protein components were concentrated to greater than 10 mg/mL using an Amicon concentrator (Millipore). Protein concentrations were calculated from their absorbances at 280 nm. The concentrated proteins were flash frozen in liquid nitrogen and stored at 80 C. These protein components were subsequently used to assemble version 7 of the system. Protein synthesis using the reconstituted in vitro protein synthesis system For proteins synthesized in batch mode, plasmid DNA was added to the reaction mixture supplemented with 4 units of RNasin (Promega). For dialysis reactions, 50 mL of the reaction mixture was placed in a microscale dialyzer (Nippon Genetics) and dialyzed against 200 mL of buffer, which contained all system components except for proteins and tRNAs. All reactions were performed at 37 C. Time course measurements of GFP synthesis were performed using a temperaturecontrolled fluorometer (Mx3005P; Agilent) and a 492/516-nm excitation/emission filter set. When necessary, in vitro protein synthesis was performed using 35S-methionine (35S-Met), and the quantity of synthesized protein was determined from the intensity of the corresponding band in autoradiograms of SDS-polyacrylamide gels. Briefly, various volumes of radiolabeled 35S-Met were spotted onto a filter paper to
We prepared three versions of the reconstituted in vitro protein synthesis system (versions 1, 2, and 7), each of which contain different component concentrations. The performance of each system was evaluated by synthesizing GFP using 200 nM RNA as a template at 37 C (Fig. 1A). Version 7 demonstrated 49-fold higher GFP fluorescence after 3 h compared with that of version 1. A list of component concentrations is provided in Table S1. Below, we describe the details of each version of the system. Version 1 was prepared according to the original study by Shimizu et al. (4), and version 2 was prepared based on two previous studies (15,16). HrpA (ATP-dependent RNA helicase) and Tig (trigger factor) were decided to be included in version 2 of the system based on a comprehensive analysis using the PURE system that indicated a beneficial role for these components in GFP synthesis (16). Additionally, the concentrations of 69 system components were systematically altered to improve the GFP yield (15), resulting in the set of concentrations used in version 2. Beginning with version 2, we first omitted chloride and glycerol from the system because an absence of these components has been shown to increase GFP yield (unpublished results). We then altered the concentration of the ribosome by threefold and increased the concentration of the other protein components, with the exception of EF-Tu (34 components in total), by approximately fourfold. Additionally, peptidyl-tRNA hydrolase (Pth) (17) was included because it was found to improve the yield by 1.2-fold. As a result of these modifications, an 8-fold increase in fluorescent GFP yield was observed for version 7 compared with that of version 2 (Fig. 1). The translation rate was determined for version 7 (Fig. 2), which synthesized GFP at a rate of 4 amino acid residues/min/RNA. We postulate that the majority of the improvement is derived from the improvement in the elongation step. This is because the increase in the EF-Tu concentration of the version 1 system to that of version 2,
FIG. 2. The rate of protein synthesis using the optimized system. (A) GFP synthesis was performed using different concentrations of RNA, and the initial rate was calculated from the band intensity in the autoradiogram. All experiments were performed in duplicate. (B) The relationship between RNA concentration and the initial rate. The translation rate was calculated from the slope of the linear regression of the data, resulting in an elongation rate of 0.96 0.06 GFP molecules/min/RNA. Because GFP is 240 residues long, the rate is equivalent to 3.84 amino acid residues/s/RNA.
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
VOL. xx, 2014 and the increase in the ribosome concentration of the version 2 system to that of version 7 were one of the most effective changes. Fig. 1 indicates that the batch reaction terminated after a few hours. The lack of an energy source, such as ATP, hinders long-term protein synthesis in cell-free systems (18). Dialysis mode, in which synthesis is performed during dialysis against a buffer containing an energy source, has been reported to be an effective strategy to prolong synthesis and to improve the yield of the synthesized proteins (19,20). Therefore, we utilized dialysis mode in our optimized system (Fig. 3A). The batch reaction yielded 0.38 mg/mL GFP, whereas the dialysis reaction yielded 3.8 mg/mL GFP after 46 h. Furthermore, 89% and 69% of the GFP produced was functional when synthesized in batch and dialysis mode, respectively. We hypothesize that the lower percentage of functional protein observed in dialysis mode is due to incomplete GFP folding at 46 h. Indeed, the amount of functional GFP increases from 30 to 46 h (Fig. 3A, closed squares). In addition, synthesized GFP was clearly visible (Fig. 3B). We also evaluated the synthesis of DHFR (18 kDa) and b-galactosidase (120 kDa) in dialysis mode and obtained final protein concentrations of 4.3 mg/mL and 4.4 mg/mL, respectively (Fig. 3C). Bands for these synthesized proteins were clearly observed on a Coomassie-stained SDS-polyacrylamide gel (Fig. 3D, Fig. S1). Although cell extract-based, cell-free protein synthesis
OPTIMIZATION OF A RECONSTITUTED CELL-FREE SYSTEM
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systems have been reported to synthesize milligram quantities of proteins (1e3), this is the first report to achieve these levels using a reconstituted (synthetic) system. The PURE system is an Escherichia coli-based reconstituted system in which protein components, with the exception of the energy regeneration reaction, are derived from E. coli. Therefore, we compared the performance of our optimized system to that of an E. coli cell. The rate of protein synthesis in vivo is 20 amino acid residues/s (21); in contrast, our system synthesized proteins at a rate of 4 amino acid residues/s, which is only 1/5 of the in vivo rate. However, the yield of the optimized in vitro system is comparable to that of an E. coli cell if the percentage of newly synthesized proteins is considered. The optimized system consisted of 2.17 mg/mL ribosomal proteins and 7.21 mg/mL additional proteins (e.g., initiation, elongation, and termination factors). This system enabled the synthesis of at most 4.4 mg/mL protein. Thus, newly synthesized protein represented 32% [¼4.4/(2.17 þ 7.21 þ 4.4)] of the entire protein in the system. When a particular protein is overexpressed in an E. coli cell, newly synthesized proteins represent at most approximately 30% of the total protein. In this regard, the optimized system has achieved the level of protein synthesis in an E. coli cell. The optimized system may be useful for various applications, including in vitro directed evolution of proteins (22,23), artificial
FIG. 3. Protein synthesis in batch and dialysis modes using DNA as a template. (A) Time course of GFP synthesis in batch and dialysis modes. Total protein concentrations were calculated from the band intensities in the autoradiogram, and the concentrations of active GFP were determined from the GFP fluorescence, assuming that the GFP synthesized and purified from E. coli was 100% active. (B) GFP synthesis using the optimized system. 1, no DNA; 2, synthesis in batch mode using version 7 of the system; and 3, synthesis in dialysis mode using version 7 of the system. (C) Time course of b-galactosidase (b-gal) and DHFR synthesis in dialysis mode. Total concentrations were calculated from the band intensities in the autoradiogram. (D) Representative data of a Coomassie-stained SDS-polyacrylamide gel. The arrows indicate the bands of the synthesized proteins. The leftmost lane represents the molecular weight marker. The result of the repeated experiment is shown in Fig. S1. A DNA concentration of 10 ng/mL was used for all experiments.
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
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cell assembly (24,25), and protein structural studies (26). Additional studies, both experimentally and theoretically using mathematical models (27,28), on the mechanism for the enhancement of the rate of synthesis and termination of the reaction may further increase protein yield, resulting in a system that produces protein yields that exceed those of E. coli cells. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.04.019. ACKNOWLEDGMENTS We thank Atsuko Uyeda, Hitomi Komai, Tomomi Sakamoto, and Ryoko Otsuki for their technical assistance. YK and TM performed the experiments, YK, NI, and TM analyzed the data, and YK, NI, TM and TY discussed the results. TM organized the project and wrote the paper. References 1. Endo, Y. and Sawasaki, T.: Cell-free expression systems for eukaryotic protein production, Curr. Opin. Biotechnol., 17, 373e380 (2006). 2. Carlson, E. D., Gan, R., Hodgman, C. E., and Jewett, M. C.: Cell-free protein synthesis: applications come of age, Biotechnol. Adv., 30, 1185e1194 (2012). 3. Schwarz, D., Dotsch, V., and Bernhard, F.: Production of membrane proteins using cell-free expression systems, Proteomics, 8, 3933e3946 (2008). 4. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T.: Cell-free translation reconstituted with purified components, Nat. Biotechnol., 19, 751e755 (2001). 5. Shimizu, Y., Kuruma, Y., Ying, B. W., Umekage, S., and Ueda, T.: Cell-free translation systems for protein engineering, FEBS J., 273, 4133e4140 (2006). 6. Kawakami, T., Murakami, H., and Suga, H.: Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids, J. Am. Chem. Soc., 130, 16861e16863 (2008). 7. Seebeck, F. P. and Szostak, J. W.: Ribosomal synthesis of dehydroalaninecontaining peptides, J. Am. Chem. Soc., 128, 7150e7151 (2006). 8. Hipolito, C. J. and Suga, H.: Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems, Curr. Opin. Chem. Biol., 16, 196e203 (2012). 9. Fujii, S., Matsuura, T., Sunami, T., Kazuta, Y., and Yomo, T.: In vitro evolution of alpha-hemolysin using a liposome display, Proc. Natl. Acad. Sci. USA, 110, 16796e16801 (2013). 10. Schlippe, Y. V., Hartman, M. C., Josephson, K., and Szostak, J. W.: In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors, J. Am. Chem. Soc., 134, 10469e10477 (2012).
J. BIOSCI. BIOENG., 11. Sobhanifar, S., Reckel, S., Junge, F., Schwarz, D., Kai, L., Karbyshev, M., Lohr, F., Bernhard, F., and Dotsch, V.: Cell-free expression and stable isotope labelling strategies for membrane proteins, J. Biomol. NMR, 46, 33e43 (2010). 12. Torizawa, T., Shimizu, M., Taoka, M., Miyano, H., and Kainosho, M.: Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol, J. Biomol. NMR, 30, 311e325 (2004). 13. Hillebrecht, J. R. and Chong, S.: A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extractbased, BMC Biotechnol., 8, 58 (2008). 14. Itoh, H., Kawazoe, Y., and Shiba, T.: Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system, J. Microbiol. Methods, 64, 241e249 (2006). 15. Matsuura, T., Kazuta, Y., Aita, T., Adachi, J., and Yomo, T.: Quantifying epistatic interactions among the components constituting the protein translation system, Mol. Syst. Biol., 5, 297 (2009). 16. Kazuta, Y., Adachi, J., Matsuura, T., Ono, N., Mori, H., and Yomo, T.: Comprehensive analysis of the effects of Escherichia coli ORFs on protein translation reaction, Mol. Cell. Proteomics, 7, 1530e1540 (2008). 17. Das, G. and Varshney, U.: Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis, Microbiology, 152, 2191e2195 (2006). 18. Kim, H. C. and Kim, D. M.: Methods for energizing cell-free protein synthesis, J. Biosci. Bioeng., 108, 1e4 (2009). 19. Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y., and Alakhov, Y. B.: A continuous cell-free translation system capable of producing polypeptides in high yield, Science, 242, 1162e1164 (1988). 20. Aoki, M., Matsuda, T., Tomo, Y., Miyata, Y., Inoue, M., Kigawa, T., and Yokoyama, S.: Automated system for high-throughput protein production using the dialysis cell-free method, Protein Expr. Purif., 68, 128e136 (2009). 21. Neidhardt, F. C. and Curtiss, R.: Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C. (1996). 22. Matsuura, T. and Yomo, T.: In vitro evolution of proteins, J. Biosci. Bioeng., 101, 449e456 (2006). 23. Pluckthun, A., Schaffitzel, C., Hanes, J., and Jermutus, L.: In vitro selection and evolution of proteins, Adv. Protein Chem., 55, 367e403 (2000). 24. Szostak, J. W., Bartel, D. P., and Luisi, P. L.: Synthesizing life, Nature, 409, 387e390 (2001). 25. Ichihashi, N., Usui, K., Kazuta, Y., Sunami, T., Matsuura, T., and Yomo, T.: Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment, Nat. Commun., 4, 2494 (2013). 26. Yokoyama, S.: Protein expression systems for structural genomics and proteomics, Curr. Opin. Chem. Biol., 7, 39e43 (2003). 27. Stogbauer, T., Windhager, L., Zimmer, R., and Radler, J. O.: Experiment and mathematical modeling of gene expression dynamics in a cell-free system, Integr. Biol. (Camb), 4, 494e501 (2012). 28. Karzbrun, E., Shin, J., Bar-Ziv, R. H., and Noireaux, V.: Coarse-grained dynamics of protein synthesis in a cell-free system, Phys. Rev. Lett., 106, 048104 (2011).
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system Yasuaki Kazuta,1 Tomoaki Matsuura,1, 2 Norikazu Ichihashi,1 and Tetsuya Yomo1, 3, 4, * Japan Science and Technology (JST), ERATO, Dynamical Microscale Reaction Environment Project, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan,1 Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan,2 Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan,3 and Graduate School of Frontier Biosciences, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan4 Received 20 February 2014; accepted 23 April 2014 Available online xxx
In this study, the amount of protein synthesized using an in vitro protein synthesis system composed of only highly purified components (the PURE system) was optimized. By varying the concentrations of each system component, we determined the component concentrations that result in the synthesis of 0.38 mg/mL green fluorescent protein (GFP) in batch mode and 3.8 mg/mL GFP in dialysis mode. In dialysis mode, protein concentrations of 4.3 and 4.4 mg/mL were synthesized for dihydrofolate reductase and b-galactosidase, respectively. Using the optimized system, the synthesized protein represented 30% (w/w) of the total protein, which is comparable to the level of overexpressed protein in Escherichia coli cells. This optimized reconstituted in vitro protein synthesis system may potentially be useful for various applications, including in vitro directed evolution of proteins, artificial cell assembly, and protein structural studies. Ó 2014, The Society for Biotechnology, Japan. All rights reserved. [Key words: In vitro protein synthesis; PURE system; Green fluorescent protein; Dialysis; Synthetic biology]
Cell-free protein synthesis enables in vitro protein synthesis without the requirement of living cells (1e3). Because the cell-free system is independent of cell growth, the synthesis of proteins that may affect cell growth is possible. Furthermore, rapid protein production is possible using a cell-free system, as protein synthesis typically requires only a few hours. Because of these favorable properties, cell-free systems have been used for various applications and basic research. Among cell-free systems, the PURE system is the only fully reconstituted system in which the minimal components for protein translation are individually purified and reconstituted in vitro (4,5). Using this reconstituted system, components can be added or omitted, and the concentration of each component can be altered. This system has enabled the efficient incorporation of unnatural amino acids into peptides (6,7). Because of the inherently low nuclease and protease activity, this system has been used for the in vitro evolution of proteins and enzymes (8e10). An additional advantage of the PURE system is the lack of amino acid scrambling, which enables nuclear magnetic resonance structural studies (11,12). However, despite its wide application, there have been few reports on the enhancement of the yield of proteins synthesized using this reconstituted in vitro protein synthesis system (13,14). In this study, we report the optimization of the PURE system, which resulted in the synthesis of greater than 4 mg/mL protein. This optimization was achieved by modifying the component
* Corresponding author at: Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita, Osaka 565-0871, Japan. Tel.: þ81 6 6879 4171; fax: þ81 6 6879 7433. E-mail address: [email protected] (T. Yomo).
concentrations and utilizing the system in dialysis mode. We have improved the protein yield of this system by including additional components and altering the component concentrations (15,16). Attempts to improve the yield of in vitro protein synthesis have been described (13,14). However, the development of a reconstituted in vitro protein synthesis system capable of synthesizing a protein at concentrations of greater than 1 mg/mL has not yet been achieved. We evaluated the performance of the optimized system using three different sets of component concentrations determined from the optimization process and demonstrate that milligram quantities of protein can be synthesized. MATERIALS AND METHODS DNA and RNA preparation The plasmid DNA used for the in vitro transcription-translation system was purified using the Qiagen Plasmid Mini Kit (Qiagen) according to the manufacturer’s instructions. Under the control of a T7 promoter, pETG5tag encoding green fluorescent protein (GFP), pET-dhfr (Gene Frontier) encoding dihydrofolate reductase (DHFR), and pET-bgal encoding bgalactosidase were used as expression plasmids. For in vitro transcription, pETG5tag was amplified using the primers T7F (50 -TAATACGACTCACTATAGGG-30 ) and T7R (50 GCTAGTTATTGCTCAGCGG-30 ) and KOD FX DNA polymerase (Toyobo, Osaka, Japan). The PCR product was purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer’s instructions. RNA was prepared from this PCR product using the MEGAscript T7 Kit (Life Technologies) and purified using an RNeasy kit (Qiagen). Component preparation Versions 1 and 2 of the system and their components were prepared as previously described (5,15). The protein components constituting version 7 of the system were prepared with additional treatment. Components, with the exception of the ribosome, were stored in stock buffer (50 mM HEPES-KOH, pH 7.6, 100 mM KCl, 10 mM MgCl2, 30% glycerol, and 7 mM 2-mercaptoethanol), and the ribosome was stored in 70S buffer (20 mM HEPESKOH, pH 7.6, 6 mM Mg(OAc)2, 30 mM KCl, and 7 mM 2-mercaptoethanol). All of these components were dialyzed against buffer A (50 mM HEPES, pH 7.6, 10 mM
1389-1723/$ e see front matter Ó 2014, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
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J. BIOSCI. BIOENG., obtain a standard curve of the relationship between the volume of 35S-Met and the signal intensity obtained in the autoradiogram. Subsequently, an appropriate volume of 35S-Met (e.g., p ml/mL) was added to the reaction mixture in which a large excess of cold Met (0.3 mM) was present. After the synthesis reaction, a portion of the sample was subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. The volume of 35S-Met (e.g., q ml/mL) was determined from the band intensity in the autoradiogram using the standard curve. The concentration of incorporated Met was calculated using the equation 0.3 (q/p) (mM), and we obtained the concentration of the synthesized proteins by dividing this value by the number of Met residues in the sequence of each synthesized protein.
Fluorescent GFP concentration ( M)
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PURE ver 1 PURE ver 2 PURE ver 7
6 4
RESULTS AND DISCUSSION
2 0
0
1
2 Time (h)
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FIG. 1. GFP synthesis using various versions of the reconstituted in vitro protein synthesis system. Time course of GFP synthesis in batch mode. RNA was used as a template at a concentration of 200 nM. GFP concentrations were determined from fluorescence intensities using the relationship between the fluorescence and concentration of GFP purified from E. coli. Purified GFP was assumed to be 100% active.
Mg(OAc)2, 100 mM potassium glutamate (K-Glu), and 7 mM 2-mercaptoethanol) using a microscale dialyzer (Nippon Genetics) with a molecular weight cut-off of 3000 Da at 4 C overnight. When the components precipitated as a result of dialysis, 3 M K-Glu was added until the pellets were dissolved. All protein components were concentrated to greater than 10 mg/mL using an Amicon concentrator (Millipore). Protein concentrations were calculated from their absorbances at 280 nm. The concentrated proteins were flash frozen in liquid nitrogen and stored at 80 C. These protein components were subsequently used to assemble version 7 of the system. Protein synthesis using the reconstituted in vitro protein synthesis system For proteins synthesized in batch mode, plasmid DNA was added to the reaction mixture supplemented with 4 units of RNasin (Promega). For dialysis reactions, 50 mL of the reaction mixture was placed in a microscale dialyzer (Nippon Genetics) and dialyzed against 200 mL of buffer, which contained all system components except for proteins and tRNAs. All reactions were performed at 37 C. Time course measurements of GFP synthesis were performed using a temperaturecontrolled fluorometer (Mx3005P; Agilent) and a 492/516-nm excitation/emission filter set. When necessary, in vitro protein synthesis was performed using 35S-methionine (35S-Met), and the quantity of synthesized protein was determined from the intensity of the corresponding band in autoradiograms of SDS-polyacrylamide gels. Briefly, various volumes of radiolabeled 35S-Met were spotted onto a filter paper to
We prepared three versions of the reconstituted in vitro protein synthesis system (versions 1, 2, and 7), each of which contain different component concentrations. The performance of each system was evaluated by synthesizing GFP using 200 nM RNA as a template at 37 C (Fig. 1A). Version 7 demonstrated 49-fold higher GFP fluorescence after 3 h compared with that of version 1. A list of component concentrations is provided in Table S1. Below, we describe the details of each version of the system. Version 1 was prepared according to the original study by Shimizu et al. (4), and version 2 was prepared based on two previous studies (15,16). HrpA (ATP-dependent RNA helicase) and Tig (trigger factor) were decided to be included in version 2 of the system based on a comprehensive analysis using the PURE system that indicated a beneficial role for these components in GFP synthesis (16). Additionally, the concentrations of 69 system components were systematically altered to improve the GFP yield (15), resulting in the set of concentrations used in version 2. Beginning with version 2, we first omitted chloride and glycerol from the system because an absence of these components has been shown to increase GFP yield (unpublished results). We then altered the concentration of the ribosome by threefold and increased the concentration of the other protein components, with the exception of EF-Tu (34 components in total), by approximately fourfold. Additionally, peptidyl-tRNA hydrolase (Pth) (17) was included because it was found to improve the yield by 1.2-fold. As a result of these modifications, an 8-fold increase in fluorescent GFP yield was observed for version 7 compared with that of version 2 (Fig. 1). The translation rate was determined for version 7 (Fig. 2), which synthesized GFP at a rate of 4 amino acid residues/min/RNA. We postulate that the majority of the improvement is derived from the improvement in the elongation step. This is because the increase in the EF-Tu concentration of the version 1 system to that of version 2,
FIG. 2. The rate of protein synthesis using the optimized system. (A) GFP synthesis was performed using different concentrations of RNA, and the initial rate was calculated from the band intensity in the autoradiogram. All experiments were performed in duplicate. (B) The relationship between RNA concentration and the initial rate. The translation rate was calculated from the slope of the linear regression of the data, resulting in an elongation rate of 0.96 0.06 GFP molecules/min/RNA. Because GFP is 240 residues long, the rate is equivalent to 3.84 amino acid residues/s/RNA.
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
VOL. xx, 2014 and the increase in the ribosome concentration of the version 2 system to that of version 7 were one of the most effective changes. Fig. 1 indicates that the batch reaction terminated after a few hours. The lack of an energy source, such as ATP, hinders long-term protein synthesis in cell-free systems (18). Dialysis mode, in which synthesis is performed during dialysis against a buffer containing an energy source, has been reported to be an effective strategy to prolong synthesis and to improve the yield of the synthesized proteins (19,20). Therefore, we utilized dialysis mode in our optimized system (Fig. 3A). The batch reaction yielded 0.38 mg/mL GFP, whereas the dialysis reaction yielded 3.8 mg/mL GFP after 46 h. Furthermore, 89% and 69% of the GFP produced was functional when synthesized in batch and dialysis mode, respectively. We hypothesize that the lower percentage of functional protein observed in dialysis mode is due to incomplete GFP folding at 46 h. Indeed, the amount of functional GFP increases from 30 to 46 h (Fig. 3A, closed squares). In addition, synthesized GFP was clearly visible (Fig. 3B). We also evaluated the synthesis of DHFR (18 kDa) and b-galactosidase (120 kDa) in dialysis mode and obtained final protein concentrations of 4.3 mg/mL and 4.4 mg/mL, respectively (Fig. 3C). Bands for these synthesized proteins were clearly observed on a Coomassie-stained SDS-polyacrylamide gel (Fig. 3D, Fig. S1). Although cell extract-based, cell-free protein synthesis
OPTIMIZATION OF A RECONSTITUTED CELL-FREE SYSTEM
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systems have been reported to synthesize milligram quantities of proteins (1e3), this is the first report to achieve these levels using a reconstituted (synthetic) system. The PURE system is an Escherichia coli-based reconstituted system in which protein components, with the exception of the energy regeneration reaction, are derived from E. coli. Therefore, we compared the performance of our optimized system to that of an E. coli cell. The rate of protein synthesis in vivo is 20 amino acid residues/s (21); in contrast, our system synthesized proteins at a rate of 4 amino acid residues/s, which is only 1/5 of the in vivo rate. However, the yield of the optimized in vitro system is comparable to that of an E. coli cell if the percentage of newly synthesized proteins is considered. The optimized system consisted of 2.17 mg/mL ribosomal proteins and 7.21 mg/mL additional proteins (e.g., initiation, elongation, and termination factors). This system enabled the synthesis of at most 4.4 mg/mL protein. Thus, newly synthesized protein represented 32% [¼4.4/(2.17 þ 7.21 þ 4.4)] of the entire protein in the system. When a particular protein is overexpressed in an E. coli cell, newly synthesized proteins represent at most approximately 30% of the total protein. In this regard, the optimized system has achieved the level of protein synthesis in an E. coli cell. The optimized system may be useful for various applications, including in vitro directed evolution of proteins (22,23), artificial
FIG. 3. Protein synthesis in batch and dialysis modes using DNA as a template. (A) Time course of GFP synthesis in batch and dialysis modes. Total protein concentrations were calculated from the band intensities in the autoradiogram, and the concentrations of active GFP were determined from the GFP fluorescence, assuming that the GFP synthesized and purified from E. coli was 100% active. (B) GFP synthesis using the optimized system. 1, no DNA; 2, synthesis in batch mode using version 7 of the system; and 3, synthesis in dialysis mode using version 7 of the system. (C) Time course of b-galactosidase (b-gal) and DHFR synthesis in dialysis mode. Total concentrations were calculated from the band intensities in the autoradiogram. (D) Representative data of a Coomassie-stained SDS-polyacrylamide gel. The arrows indicate the bands of the synthesized proteins. The leftmost lane represents the molecular weight marker. The result of the repeated experiment is shown in Fig. S1. A DNA concentration of 10 ng/mL was used for all experiments.
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019
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cell assembly (24,25), and protein structural studies (26). Additional studies, both experimentally and theoretically using mathematical models (27,28), on the mechanism for the enhancement of the rate of synthesis and termination of the reaction may further increase protein yield, resulting in a system that produces protein yields that exceed those of E. coli cells. Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jbiosc.2014.04.019. ACKNOWLEDGMENTS We thank Atsuko Uyeda, Hitomi Komai, Tomomi Sakamoto, and Ryoko Otsuki for their technical assistance. YK and TM performed the experiments, YK, NI, and TM analyzed the data, and YK, NI, TM and TY discussed the results. TM organized the project and wrote the paper. References 1. Endo, Y. and Sawasaki, T.: Cell-free expression systems for eukaryotic protein production, Curr. Opin. Biotechnol., 17, 373e380 (2006). 2. Carlson, E. D., Gan, R., Hodgman, C. E., and Jewett, M. C.: Cell-free protein synthesis: applications come of age, Biotechnol. Adv., 30, 1185e1194 (2012). 3. Schwarz, D., Dotsch, V., and Bernhard, F.: Production of membrane proteins using cell-free expression systems, Proteomics, 8, 3933e3946 (2008). 4. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T.: Cell-free translation reconstituted with purified components, Nat. Biotechnol., 19, 751e755 (2001). 5. Shimizu, Y., Kuruma, Y., Ying, B. W., Umekage, S., and Ueda, T.: Cell-free translation systems for protein engineering, FEBS J., 273, 4133e4140 (2006). 6. Kawakami, T., Murakami, H., and Suga, H.: Ribosomal synthesis of polypeptoids and peptoid-peptide hybrids, J. Am. Chem. Soc., 130, 16861e16863 (2008). 7. Seebeck, F. P. and Szostak, J. W.: Ribosomal synthesis of dehydroalaninecontaining peptides, J. Am. Chem. Soc., 128, 7150e7151 (2006). 8. Hipolito, C. J. and Suga, H.: Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems, Curr. Opin. Chem. Biol., 16, 196e203 (2012). 9. Fujii, S., Matsuura, T., Sunami, T., Kazuta, Y., and Yomo, T.: In vitro evolution of alpha-hemolysin using a liposome display, Proc. Natl. Acad. Sci. USA, 110, 16796e16801 (2013). 10. Schlippe, Y. V., Hartman, M. C., Josephson, K., and Szostak, J. W.: In vitro selection of highly modified cyclic peptides that act as tight binding inhibitors, J. Am. Chem. Soc., 134, 10469e10477 (2012).
J. BIOSCI. BIOENG., 11. Sobhanifar, S., Reckel, S., Junge, F., Schwarz, D., Kai, L., Karbyshev, M., Lohr, F., Bernhard, F., and Dotsch, V.: Cell-free expression and stable isotope labelling strategies for membrane proteins, J. Biomol. NMR, 46, 33e43 (2010). 12. Torizawa, T., Shimizu, M., Taoka, M., Miyano, H., and Kainosho, M.: Efficient production of isotopically labeled proteins by cell-free synthesis: a practical protocol, J. Biomol. NMR, 30, 311e325 (2004). 13. Hillebrecht, J. R. and Chong, S.: A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extractbased, BMC Biotechnol., 8, 58 (2008). 14. Itoh, H., Kawazoe, Y., and Shiba, T.: Enhancement of protein synthesis by an inorganic polyphosphate in an E. coli cell-free system, J. Microbiol. Methods, 64, 241e249 (2006). 15. Matsuura, T., Kazuta, Y., Aita, T., Adachi, J., and Yomo, T.: Quantifying epistatic interactions among the components constituting the protein translation system, Mol. Syst. Biol., 5, 297 (2009). 16. Kazuta, Y., Adachi, J., Matsuura, T., Ono, N., Mori, H., and Yomo, T.: Comprehensive analysis of the effects of Escherichia coli ORFs on protein translation reaction, Mol. Cell. Proteomics, 7, 1530e1540 (2008). 17. Das, G. and Varshney, U.: Peptidyl-tRNA hydrolase and its critical role in protein biosynthesis, Microbiology, 152, 2191e2195 (2006). 18. Kim, H. C. and Kim, D. M.: Methods for energizing cell-free protein synthesis, J. Biosci. Bioeng., 108, 1e4 (2009). 19. Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Y., and Alakhov, Y. B.: A continuous cell-free translation system capable of producing polypeptides in high yield, Science, 242, 1162e1164 (1988). 20. Aoki, M., Matsuda, T., Tomo, Y., Miyata, Y., Inoue, M., Kigawa, T., and Yokoyama, S.: Automated system for high-throughput protein production using the dialysis cell-free method, Protein Expr. Purif., 68, 128e136 (2009). 21. Neidhardt, F. C. and Curtiss, R.: Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C. (1996). 22. Matsuura, T. and Yomo, T.: In vitro evolution of proteins, J. Biosci. Bioeng., 101, 449e456 (2006). 23. Pluckthun, A., Schaffitzel, C., Hanes, J., and Jermutus, L.: In vitro selection and evolution of proteins, Adv. Protein Chem., 55, 367e403 (2000). 24. Szostak, J. W., Bartel, D. P., and Luisi, P. L.: Synthesizing life, Nature, 409, 387e390 (2001). 25. Ichihashi, N., Usui, K., Kazuta, Y., Sunami, T., Matsuura, T., and Yomo, T.: Darwinian evolution in a translation-coupled RNA replication system within a cell-like compartment, Nat. Commun., 4, 2494 (2013). 26. Yokoyama, S.: Protein expression systems for structural genomics and proteomics, Curr. Opin. Chem. Biol., 7, 39e43 (2003). 27. Stogbauer, T., Windhager, L., Zimmer, R., and Radler, J. O.: Experiment and mathematical modeling of gene expression dynamics in a cell-free system, Integr. Biol. (Camb), 4, 494e501 (2012). 28. Karzbrun, E., Shin, J., Bar-Ziv, R. H., and Noireaux, V.: Coarse-grained dynamics of protein synthesis in a cell-free system, Phys. Rev. Lett., 106, 048104 (2011).
Please cite this article in press as: Kazuta, Y., et al., Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system, J. Biosci. Bioeng., (2014), http://dx.doi.org/10.1016/j.jbiosc.2014.04.019