Overview of Cell‐Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

Shaorong Chong1

1 New England Biolabs, Ipswich, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.30
DOI:  10.1002/0471142727.mb1630s108
Online Posting Date:  October, 2014
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Abstract

During the early days of molecular biology, cell‐free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell‐free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell‐free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell‐free system has certain advantages and disadvantages, the diversity of the cell‐free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post‐genomic era, cell‐free protein synthesis has rapidly become the preferred approach for high‐throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell‐free protein synthesis and describes key advances in modern cell‐free systems, practical differences between widely used commercial cell‐free systems, and applications of this important technology. Curr. Protoc. Mol. Biol. 108:16.30.1‐16.30.11. © 2014 by John Wiley & Sons, Inc.

Keywords: cell‐free protein synthesis; high throughput; unnatural amino acid; isotope labeling; in vitro protein evolution; artificial cell

     
 
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Table of Contents

  • Introduction
  • Early History of Cell‐Free Protein Synthesis
  • Landmark Advances in the Development of Cell‐Free Protein Synthesis Systems
  • Use of Commercially Available Cell‐Free Protein Synthesis Systems
  • Application of Cell‐Free Protein Synthesis
  • Conclusion
  • Tables
     
 
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Materials

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Literature Cited

Literature Cited
  Anderson, C.W., Straus, J.W., and Dudock, B.S. 1983. Preparation of a cell‐free protein‐synthesizing system from wheat germ. Methods Enzymol. 101:635‐644.
  Aoki, M., Matsuda, T., Tomo, Y., Miyata, Y., Inoue, M., Kigawa, T., and Yokoyama, S. 2009. Automated system for high‐throughput protein production using the dialysis cell‐free method. Protein Expr. Purif. 68:128‐136.
  Asahara, H. and Chong, S. 2010. In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme. Nucleic Acids Res. 38:e141.
  Bank, A. and Marks, P.A. 1966. Protein synthesis in a cell free human reticulocyte system: Ribosome function in thalassemia. J. Clin. Invest. 45:330‐336.
  Boder, E.T. and Wittrup, K.D. 1997. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15:553‐557.
  Carlson, E.D., Gan, R., Hodgman, C.E., and Jewett, M.C. 2012. Cell‐free protein synthesis: Applications come of age. Biotechnol. Adv. 30:1185‐1194.
  Chen, H.Z. and Zubay, G. 1983. Prokaryotic coupled transcription‐translation. Methods Enzymol. 101:674‐690.
  Courtois, F., Olguin, L.F., Whyte, G., Bratton, D., Huck, W.T., Abell, C., and Hollfelder, F. 2008. An integrated device for monitoring time‐dependent in vitro expression from single genes in picolitre droplets. Chembiochem 9:439‐446.
  Craig, D., Howell, M.T., Gibbs, C.L., Hunt, T., and Jackson, R.J. 1992. Plasmid cDNA‐directed protein synthesis in a coupled eukaryotic in vitro transcription‐translation system. Nucleic Acids Res. 20:4987‐4995.
  Crick, F. 1970. Central dogma of molecular biology. Nature 227:561‐563.
  Derda, R., Tang, S.K., Li, S.C., Ng, S., Matochko, W., and Jafari, M.R. 2011. Diversity of phage‐displayed libraries of peptides during panning and amplification. Molecules 16:1776‐1803.
  Dittrich, P.S., Jahnz, M., and Schwille, P. 2005. A new embedded process for compartmentalized cell‐free protein expression and on‐line detection in microfluidic devices. Chembiochem 6:811‐814.
  Dougherty, M.J. and Arnold, F.H. 2009. Directed evolution: New parts and optimized function. Curr. Opin. Biotechnol. 20:486‐491.
  Erickson, A.H. and Blobel, G. 1983. Cell‐free translation of messenger RNA in a wheat germ system. Methods Enzymol. 96:38‐50.
  Ezure, T., Suzuki, T., Higashide, S., Shintani, E., Endo, K., Kobayashi, S., Shikata, M., Ito, M., Tanimizu, K., and Nishimura, O. 2006. Cell‐free protein synthesis system prepared from insect cells by freeze‐thawing. Biotechnol. Prog. 22:1570‐1577.
  Ezure, T., Suzuki, T., Shikata, M., Ito, M., Ando, E., Utsumi, T., Nishimura, O., and Tsunasawa, S. 2010. Development of an insect cell‐free system. Curr. Pharm. Biotechnol. 11:279‐284.
  Fallah‐Araghi, A., Baret, J.C., Ryckelynck, M., and Griffiths, A.D. 2012. A completely in vitro ultrahigh‐throughput droplet‐based microfluidic screening system for protein engineering and directed evolution. Lab. Chip 12:882‐891.
  Farinas, E.T., Bulter, T., and Arnold, F.H. 2001. Directed enzyme evolution. Curr. Opin. Biotechnol. 12:545‐551.
  Festa, F., Rollins, S.M., Vattem, K., Hathaway, M., Lorenz, P., Mendoza, E.A., Yu, X., Qiu, J., Kilmer, G., Jensen, P., Webb, B., Ryan, E.T., and LaBaer, J. 2013. Robust microarray production of freshly expressed proteins in a human milieu. Proteomics Clin. Appl. 7:372‐377.
  Forster, A.C. and Church, G.M. 2006. Towards synthesis of a minimal cell. Mol. Syst. Biol. 2:45.
  Forster, A.C., Tan, Z., Nalam, M.N., Lin, H., Qu, H., Cornish, V.W., and Blacklow, S.C. 2003. Programming peptidomimetic syntheses by translating genetic codes designed de novo. Proc. Natl. Acad. Sci. U.S.A. 100:6353‐6357.
  Forster, A.C., Cornish, V.W., and Blacklow, S.C. 2004. Pure translation display. Anal. Biochem. 333:358‐364.
  Gai, S.A. and Wittrup, K.D. 2007. Yeast surface display for protein engineering and characterization. Curr. Opin. Struct. Biol. 17:467‐473.
  Gale, E.F. and Folkes, J.P. 1954. Effect of nucleic acids on protein synthesis and amino‐acid incorporation in disrupted staphylococcal cells. Nature 173:1223‐1227.
  Ganoza, M.C., Cunningham, C., and Green, R.M. 1985. Isolation and point of action of a factor from Escherichia coli required to reconstruct translation. Proc. Natl. Acad. Sci. U.S.A. 82:1648‐1652.
  Gesteland, R.F. 1966. Isolation and characterization of ribonuclease I mutants of Escherichia coli. J. Mol. Biol. 16:67‐84.
  Gil, R., Silva, F.J., Pereto, J., and Moya, A. 2004. Determination of the core of a minimal bacterial gene set. Microbiol. Mol. Biol. Rev. 68:518‐537.
  Golynskiy, M.V., Haugner, J.C., 3rd, Morelli, A., Morrone, D., and Seelig, B. 2013. In vitro evolution of enzymes. Methods Mol. Biol. 978:73‐92.
  Griffiths, A.D. and Tawfik, D.S. 2000. Man‐made enzymes–from design to in vitro compartmentalisation. Curr. Opin. Biotechnol. 11:338‐353.
  Griffiths, A.D. and Tawfik, D.S. 2006. Miniaturising the laboratory in emulsion droplets. Trends Biotechnol. 24:395‐402.
  Hanes, J. and Pluckthun, A. 1997. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. U.S.A. 94:4937‐4942.
  Hanes, J., Jermutus, L., Schaffitzel, C., and Pluckthun, A. 1999. Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems. FEBS Lett. 450:105‐110.
  Hartman, M.C., Josephson, K., and Szostak, J.W. 2006. Enzymatic aminoacylation of tRNA with unnatural amino acids. Proc. Natl. Acad. Sci. U.S.A. 103:4356‐4361.
  He, M. and Taussig, M.J. 1997. Antibody‐ribosome‐mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res. 25:5132‐5134.
  He, M. and Taussig, M.J. 2008. Production of protein arrays by cell‐free systems. Methods Mol. Biol. 484:207‐215.
  He, M., Stoevesandt, O., and Taussig, M.J. 2008. In situ synthesis of protein arrays. Curr. Opin. Biotechnol. 19:4‐9.
  Hibbert, E.G. and Dalby, P.A. 2005. Directed evolution strategies for improved enzymatic performance. Microb. Cell Fact. 4:29.
  Hipolito, C.J. and Suga, H. 2012. Ribosomal production and in vitro selection of natural product‐like peptidomimetics: The FIT and RaPID systems. Curr. Opin. Chem. Biol. 16:196‐203.
  Hoagland, M.B., Keller, E.B., and Zamecnik, P.C. 1956. Enzymatic carboxyl activation of amino acids. J. Biol. Chem. 218:345‐358.
  Jackson, R.J. and Hunt, T. 1983. Preparation and use of nuclease‐treated rabbit reticulocyte lysates for the translation of eukaryotic messenger RNA. Methods Enzymol. 96:50‐74.
  Jewett, M.C. and Swartz, J.R. 2004a. Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis. Biotechnol. Bioeng. 86:19‐26.
  Jewett, M.C. and Swartz, J.R. 2004b. Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm. Biotechnol. Bioeng. 87:465‐472.
  Jewett, M.C. and Forster, A.C. 2010. Update on designing and building minimal cells. Curr. Opin. Biotechnol. 21:697‐703.
  Jewett, M.C., Calhoun, K.A., Voloshin, A., Wuu, J.J., and Swartz, J.R. 2008. An integrated cell‐free metabolic platform for protein production and synthetic biology. Mol. Syst. Biol. 4:220.
  Josephson, K., Hartman, M.C., and Szostak, J.W. 2005. Ribosomal synthesis of unnatural peptides. J. Am. Chem. Soc. 127:11727‐11735.
  Josephson, K., Ricardo, A., and Szostak, J.W. 2013. mRNA display: From basic principles to macrocycle drug discovery. Drug. Discov. Today. 19:388‐399.
  Kawasaki, T., Gouda, M.D., Sawasaki, T., Takai, K., and Endo, Y. 2003. Efficient synthesis of a disulfide‐containing protein through a batch cell‐free system from wheat germ. Eur. J. Biochem. 270:4780‐4786.
  Keller, E.B. and Littlefield, J.W. 1957. Incorporation of C14‐amino acids into ribonucleoprotein particles from the Ehrlich mouse ascites tumor. J. Biol. Chem. 224:13‐30.
  Kim, T.W., Keum, J.W., Oh, I.S., Choi, C.Y., Park, C.G., and Kim, D.M. 2006. Simple procedures for the construction of a robust and cost‐effective cell‐free protein synthesis system. J. Biotechnol. 126:554‐561.
  Koonin, E.V., Mushegian, A.R., and Rudd, K.E. 1996. Sequencing and analysis of bacterial genomes. Curr. Biol. 6:404‐416.
  Krieg, P.A. and Melton, D.A. 1987. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 155:397‐415.
  Kung, H.F., Redfield, B., Treadwell, B.V., Eskin, B., Spears, C., and Weissbach, H. 1977. DNA‐directed in vitro synthesis of beta‐galactosidase. Studies with purified factors. J. Biol. Chem. 252:6889‐6894.
  Kuruma, Y., Nishiyama, K., Shimizu, Y., Muller, M., and Ueda, T. 2005. Development of a minimal cell‐free translation system for the synthesis of presecretory and integral membrane proteins. Biotechnol. Prog. 21:1243‐1251.
  Lamborg, M.R. and Zamecnik, P.C. 1960. Amino acid incorporation into protein by extracts of E. coli. Biochim. Biophys. Acta 42:206‐211.
  Littlefield, J.W., Keller, E.B., Gross, J., and Zamecnik, P.C. 1955. Studies on cytoplasmic ribonucleoprotein particles from the liver of the rat. J. Biol. Chem. 217:111‐123.
  Liu, D.V., Zawada, J.F., and Swartz, J.R. 2005. Streamlining Escherichia coli S30 extract preparation for economical cell‐free protein synthesis. Biotechnol. Prog. 21:460‐465.
  Lu, W.C. and Ellington, A.D. 2013. In vitro selection of proteins via emulsion compartments. Methods 60:75‐80.
  MacBeath, G. and Schreiber, S.L. 2000. Printing proteins as microarrays for high‐throughput function determination. Science 289:1760‐1763.
  Madin, K., Sawasaki, T., Ogasawara, T., and Endo, Y. 2000. A highly efficient and robust cell‐free protein synthesis system prepared from wheat embryos: Plants apparently contain a suicide system directed at ribosomes. Proc. Natl. Acad. Sci. U.S.A. 97:559‐564.
  Marcus, A. and Feeley, J. 1966. Ribosome activation and polysome formation in vitro: Requirement for ATP. Proc. Natl. Acad. Sci. U.S.A. 56:1770‐1777.
  Matthaei, J.H. and Nirenberg, M.W. 1961. Characteristics and stabilization of DNAase‐sensitive protein synthesis in E. coli extracts. Proc. Natl. Acad. Sci. U.S.A. 47:1580‐1588.
  Michel‐Reydellet, N., Calhoun, K., and Swartz, J. 2004. Amino acid stabilization for cell‐free protein synthesis by modification of the Escherichia coli genome. Metab. Eng. 6:197‐203.
  Miersch, S. and LaBaer, J. 2011. Nucleic Acid programmable protein arrays: Versatile tools for array‐based functional protein studies. Curr. Protoc. Protein Sci. 64:27.2.1‐27.2.26.
  Mikami, S., Kobayashi, T., Yokoyama, S., and Imataka, H. 2006a. A hybridoma‐based in vitro translation system that efficiently synthesizes glycoproteins. J. Biotechnol. 127:65‐78.
  Mikami, S., Masutani, M., Sonenberg, N., Yokoyama, S., and Imataka, H. 2006b. An efficient mammalian cell‐free translation system supplemented with translation factors. Protein Expr. Purif. 46:348‐357.
  Mikami, S., Kobayashi, T., and Imataka, H. 2010. Cell‐free protein synthesis systems with extracts from cultured human cells. Methods Mol. Biol. 607:43‐52.
  Murakami, H., Ohta, A., Ashigai, H., and Suga, H. 2006. A highly flexible tRNA acylation method for non‐natural polypeptide synthesis. Nat. Methods 3:357‐359.
  Nevin, D.E. and Pratt, J.M. 1991. A coupled in vitro transcription‐translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter. FEBS Lett. 291:259‐263.
  Nirenberg, M. 2004. Historical review: Deciphering the genetic code–a personal account. Trends Biochem. Sci. 29:46‐54.
  Nirenberg, M.W. and Matthaei, J.H. 1961. The dependence of cell‐free protein synthesis in E. coli upon naturally occurring or synthetic polyribonucleotides. Proc. Natl. Acad. Sci. U.S.A. 47:1588‐1602.
  Noireaux, V. and Libchaber, A. 2004. A vesicle bioreactor as a step toward an artificial cell assembly. Proc. Natl. Acad. Sci. U.S.A. 101:17669‐17674.
  Noireaux, V., Maeda, Y.T., and Libchaber, A. 2011. Development of an artificial cell, from self‐organization to computation and self‐reproduction. Proc. Natl. Acad. Sci. U.S.A. 108:3473‐3480.
  Noren, C.J., Anthony‐Cahill, S.J., Griffith, M.C., and Schultz, P.G. 1989. A general method for site‐specific incorporation of unnatural amino acids into proteins. Science 244:182‐188.
  Nozawa, A. and Tozawa, Y. 2014. Modifications of wheat germ cell‐free system for functional proteomics of plant membrane proteins. Methods Mol. Biol. 1072:259‐272.
  Nozawa, A., Ogasawara, T., Matsunaga, S., Iwasaki, T., Sawasaki, T., and Endo, Y. 2011. Production and partial purification of membrane proteins using a liposome‐supplemented wheat cell‐free translation system. BMC Biotechnol. 11:35.
  Oberholzer, T., Wick, R., Luisi, P.L., and Biebricher, C.K. 1995. Enzymatic RNA replication in self‐reproducing vesicles: An approach to a minimal cell. Biochem. Biophys. Res. Commun. 207:250‐257.
  Pelham, H.R. and Jackson, R.J. 1976. An efficient mRNA‐dependent translation system from reticulocyte lysates. Eur. J. Biochem. 67:247‐256.
  Pereira de Souza, T., Stano, P., and Luisi, P.L. 2009. The minimal size of liposome‐based model cells brings about a remarkably enhanced entrapment and protein synthesis. Chembiochem 10:1056‐1063.
  Pluckthun, A. 2012. Ribosome display: A perspective. Methods Mol. Biol. 805:3‐28.
  Pratt, J.M. 1984. Coupled transcription‐translation in prokaryotic cell‐free systems. In Transcription and Translation: A Practical Approach (B.D. Hames and S.J. Higgins, eds.) pp. 179‐209. IRL Press, Oxford, United Kingdom.
  Roberts, B.E. and Paterson, B.M. 1973. Efficient translation of tobacco mosaic virus RNA and rabbit globin 9S RNA in a cell‐free system from commercial wheat germ. Proc. Natl. Acad. Sci. U.S.A. 70:2330‐2334.
  Roberts, R.W. and Szostak, J.W. 1997. RNA‐peptide fusions for the in vitro selection of peptides and proteins. Proc. Natl. Acad. Sci. U.S.A. 94:12297‐12302.
  Romero, P.A. and Arnold, F.H. 2009. Exploring protein fitness landscapes by directed evolution. Nat. Rev. Mol. Cell Biol. 10:866‐876.
  Ryabova, L.A., Morozov, I., and Spirin, A.S. 1998. Continuous‐flow cell‐free translation, transcription‐translation, and replication‐translation systems. Methods Mol. Biol. 77:179‐193.
  Sawasaki, T., Gouda, M.D., Kawasaki, T., Tsuboi, T., Tozawa, Y., Takai, K., and Endo, Y. 2005. The wheat germ cell‐free expression system: Methods for high‐throughput materialization of genetic information. Methods Mol. Biol. 310:131‐144.
  Sawasaki, T., Morishita, R., Gouda, M.D., and Endo, Y. 2007. Methods for high‐throughput materialization of genetic information based on wheat germ cell‐free expression system. Methods Mol. Biol. 375:95‐106.
  Schneider, B., Junge, F., Shirokov, V.A., Durst, F., Schwarz, D., Dotsch, V., and Bernhard, F. 2010. Membrane protein expression in cell‐free systems. Methods Mol. Biol. 601:165‐186.
  Schweet, R., Lamfrom, H., and Allen, E. 1958. The synthesis of hemoglobin in a cell‐free system. Proc. Natl. Acad. Sci. U.S.A. 44:1029‐1035.
  Shimizu, Y. and Ueda, T. 2010. PURE technology. Methods Mol. Biol. 607:11‐21.
  Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., and Ueda, T. 2001. Cell‐free translation reconstituted with purified components. Nat. Biotechnol. 19:751‐755.
  Shimizu, Y., Kanamori, T., and Ueda, T. 2005. Protein synthesis by pure translation systems. Methods 36:299‐304.
  Shin, J. and Noireaux, V. 2012. An E. coli cell‐free expression toolbox: Application to synthetic gene circuits and artificial cells. ACS Synth. Biol. 1:29‐41.
  Shirokov, V.A., Kommer, A., Kolb, V.A., and Spirin, A.S. 2007. Continuous‐exchange protein‐synthesizing systems. Methods Mol. Biol. 375:19‐55.
  Sidhu, S.S. and Koide, S. 2007. Phage display for engineering and analyzing protein interaction interfaces. Curr. Opin. Struct. Biol. 17:481‐487.
  Singh‐Blom, A., Hughes, R.A., and Ellington, A.D. 2013. Residue‐specific incorporation of unnatural amino acids into proteins in vitro and in vivo. Methods Mol. Biol. 978:93‐114.
  Smith, G.P. 1985. Filamentous fusion phage: Novel expression vectors that display cloned antigens on the virion surface. Science 228:1315‐1317.
  Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y., and Alakhov, Y.B. 1988. A continuous cell‐free translation system capable of producing polypeptides in high yield. Science 242:1162‐1164.
  Strasser, B.J. 2006. A world in one dimension: Linus Pauling, Francis Crick and the central dogma of molecular biology. Hist. Philos. Life Sci. 28:491‐512.
  Suzuki, T., Ito, M., Ezure, T., Shikata, M., Ando, E., Utsumi, T., Tsunasawa, S., and Nishimura, O. 2007. Protein prenylation in an insect cell‐free protein synthesis system and identification of products by mass spectrometry. Proteomics 7:1942‐1950.
  Suzuki, T., Moriya, K., Nagatoshi, K., Ota, Y., Ezure, T., Ando, E., Tsunasawa, S., and Utsumi, T. 2010. Strategy for comprehensive identification of human N‐myristoylated proteins using an insect cell‐free protein synthesis system. Proteomics 10:1780‐1793.
  Swartz, J.R., Jewett, M.C., and Woodrow, K.A. 2004. Cell‐free protein synthesis with prokaryotic combined transcription‐translation. Methods Mol. Biol. 267:169‐182.
  Takahashi, F., Ebihara, T., Mie, M., Yanagida, Y., Endo, Y., Kobatake, E., and Aizawa, M. 2002. Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads. FEBS Lett. 514:106‐110.
  Takahashi, T.T. and Roberts, R.W. 2009. In vitro selection of protein and peptide libraries using mRNA display. Methods Mol. Biol. 535:293‐314.
  Tarui, H., Imanishi, S., and Hara, T. 2000. A novel cell‐free translation/glycosylation system prepared from insect cells. J. Biosci. Bioeng. 90:508‐514.
  Tarui, H., Murata, M., Tani, I., Imanishi, S., Nishikawa, S., and Hara, T. 2001. Establishment and characterization of cell‐free translation/glycosylation in insect cell (Spodoptera frugiperda 21) extract prepared with high pressure treatment. Appl. Microbiol. Biotechnol. 55:446‐453.
  Ueda, T., Kanamori, T., and Ohashi, H. 2010. Ribosome display with the PURE technology. Methods Mol. Biol. 607:219‐225.
  Villemagne, D., Jackson, R., and Douthwaite, J.A. 2006. Highly efficient ribosome display selection by use of purified components for in vitro translation. J. Immunol. Methods 313:140‐148.
  Vinarov, D.A. and Markley, J.L. 2005. High‐throughput automated platform for nuclear magnetic resonance‐based structural proteomics. Exp. Rev. Proteomics 2:49‐55.
  Wang, L., Brock, A., Herberich, B., and Schultz, P.G. 2001. Expanding the genetic code of Escherichia coli. Science 292:498‐500.
  Watts, R.E. and Forster, A.C. 2012. Update on pure translation display with unnatural amino acid incorporation. Methods Mol. Biol. 805:349‐365.
  Yamauchi, S., Fusada, N., Hayashi, H., Utsumi, T., Uozumi, N., Endo, Y., and Tozawa, Y. 2010. The consensus motif for N‐myristoylation of plant proteins in a wheat germ cell‐free translation system. FEBS J. 277:3596‐3607.
  Yang, H.L., Ivashkiv, L., Chen, H.Z., Zubay, G., and Cashel, M. 1980. Cell‐free coupled transcription‐translation system for investigation of linear DNA segments. Proc. Natl. Acad. Sci. U.S.A. 77:7029‐7033.
  Young, T.S. and Schultz, P.G. 2010. Beyond the canonical 20 amino acids: Expanding the genetic lexicon. J. Biol. Chem. 285:11039‐11044.
  Zamecnik, P.C., Frantz, I.D. Jr. et al. 1948. Incorporation in vitro of radioactive carbon from carboxyl‐labeled DL‐alanine and glycine into proteins of normal and malignant rat livers. J. Biol. Chem. 175:299‐314.
  Zawada, J. and Swartz, J. 2005. Maintaining rapid growth in moderate‐density Escherichia coli fermentations. Biotechnol. Bioeng. 89:407‐415.
  Zawada, J. and Swartz, J. 2006. Effects of growth rate on cell extract performance in cell‐free protein synthesis. Biotechnol. Bioeng. 94:618‐624.
  Zawada, J.F., Yin, G., Steiner, A.R., Yang, J., Naresh, A., Roy, S.M., Gold, D.S., Heinsohn, H.G., and Murray, C.J. 2011. Microscale to manufacturing scale‐up of cell‐free cytokine production—a new approach for shortening protein production development timelines. Biotechnol. Bioeng. 108:1570‐1578.
  Zhou, Y., Asahara, H., Gaucher, E.A., and Chong, S. 2012. Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components. Nucleic Acids Res. 40:7932‐7945.
  Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R., Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., and Snyder, M. 2001. Global analysis of protein activities using proteome chips. Science 293:2101‐2105.
  Zubay, G. 1973. In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7:267‐287.
Key References
  Spirin, A.S. and Swartz, J.R. (eds.). 2008. Cell‐free Protein Synthesis Methods and Protocols. Wiley‐VCH, Weinheim, Germany.
  Edited by two leading figures in the field, this book provides in‐depth reviews of almost every aspect of cell‐free protein synthesis, contributed by experts in different cell‐free systems.
  Nirenberg, M. 2004. See above.
  Marshall Nirenberg gave a vivid personal account of how his group used cell‐free protein synthesis to decipher the genetic code.
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