Skip to main content
SpringerLink
Log in

Continuous-Exchange Protein-Synthesizing Systems

  • Protocol
In Vitro Transcription and Translation Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 375))

Summary

Protein synthesis in cell-free systems is an emerging technology already competing with in vivo expression methods. In this chapter the basic principles of continuous-exchange protein synthesizing systems, and protocols for Escherichia coli and wheat germ translation and transcription-translation systems are described. The ways to improve substrate supply in cell-free systems and mRNA design for eukaryotic system are discussed. Correct folding of the synthesized protein is demonstrated and discussed in detail.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. 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.

    Article  CAS  PubMed  Google Scholar 

  2. Baranov, V. I., Morozov, I. Y., Ortlepp, S. A., and Spirin, A. S. (1989) Gene expression in a cell-free system on the preparative scale. Gene 84, 463–466.

    Article  CAS  PubMed  Google Scholar 

  3. Baranov, V. I. and Spirin, A. S. (1993) Gene expression in cell-free systems on preparative scale. Methods Enzymol. 217, 123–142.

    Article  CAS  PubMed  Google Scholar 

  4. Alakhov, Y. B., Baranov, V. I., Ovodov, S. Y., Ryabova, L. A., and Spirin, A. S. (1995) Method of preparing polypeptides in cell-free translation system. United States Patent no. 5,478,730.

    Google Scholar 

  5. Chekulayeva, M. N., Kurnasov, O. V., Shirokov, V. A., and Spirin, A. S. (2001) Continuous-exchange cell-free protein-synthesizing system: Synthesis of HIV-1 antigen Nef. Biochem. Biophys. Res. Commun. 280, 914–917.

    Article  CAS  PubMed  Google Scholar 

  6. Martemyanov, K. A., Shirokov, V. A., Kurnasov, O. V., Gudkov, A. T., and Spirin, A. S. (2001) Cell-free production of biologically active polypeptides: Application to the synthesis of antibacterial peptide Cecropin. Protein Expr. Purif. 21, 456–461.

    Article  CAS  PubMed  Google Scholar 

  7. Shirokov, V. A., Simonenko, P. N., Biryukov, S. A., and Spirin, A. S. (2002) Continuous-flow and continuous-exchange cell-free translation systems and reactors, in Cell-Free Translation Systems, (Spirin, A. S., ed.), Springer-Verlag, Berlin, Germany, pp. 91–107.

    Google Scholar 

  8. Spirin, A. S. (2004) High-throughput cell-free systems for synthesis of functionally active proteins. Trends Biotech. 22, 538–545.

    Article  CAS  Google Scholar 

  9. Kim, D.-M. and Choi, C.-Y. (1996) A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane. Biotechnol. Prog. 12, 645–649.

    Article  CAS  PubMed  Google Scholar 

  10. Davis, J., Thompson, D., and Beckler, G. S. (1996) Large scale dialysis cell-free system. Promega Notes Magazine 56, 14–21.

    Google Scholar 

  11. Kigawa, T., Yabuki, T., Yoshida, Y., et al. (1999) Cell-free production and stable-isotope labelling of milligram quantities of proteins. FEBS Lett. 442, 15–19.

    Article  CAS  PubMed  Google Scholar 

  12. 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. USA 97, 559–564.

    Article  CAS  PubMed  Google Scholar 

  13. Martin, G. A., Kawaguchi, R., Lam, Y., DeGiovanni, A., Fukushima, M., and Mutter, W. (2001) High-yield, in vitro protein expression using a continuous-exchange, coupled transcription/translation system. BioTechniques 31, 948–953.

    CAS  PubMed  Google Scholar 

  14. Sawasaki, T., Ogasawara, T., Morishita, R., and Endo, Y. (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc. Natl. Acad. Sci. USA 99, 14,652–14,657.

    Article  CAS  PubMed  Google Scholar 

  15. Endo, Y. and Sawasaki, T. (2004) High-throughput, genome scale protein production method based on the wheat germ cell-free expression system. J. Struct. Funct. Genomics 5, 45–57.

    Article  CAS  PubMed  Google Scholar 

  16. Yokoyama, S. (2003) Protein expression systems for structural genomics and proteomics. Curr. Op. Chem. Biol. 7, 39–43.

    Article  CAS  Google Scholar 

  17. Betton, J.-M. (2003) Rapid translation system (RTS): a promising alternative for recombinant protein production. Current Protein and Peptide Science 4, 73–80.

    Article  CAS  PubMed  Google Scholar 

  18. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  19. Pokrovskaya, I. D. and Gurevich, V. V. (1994) In vitro transcription: preparative RNA yields in analytical reactions. Anal. Biochem. 220, 420–423.

    Article  CAS  PubMed  Google Scholar 

  20. Zubay, G. (1973) In vitro synthesis of protein in microbial systems. Annu. Rev. Genet. 7, 267–287.

    Article  CAS  PubMed  Google Scholar 

  21. Erickson, A. H. and Blobel, G. (1983) Cell-free translation of messenger RNA in a wheat germ system. Methods Enzymol. 96, 38–50.

    Article  CAS  PubMed  Google Scholar 

  22. 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.

    Article  CAS  PubMed  Google Scholar 

  23. Kim, D. M., Kigawa, T., Choi, C. Y., and Yokoyama, S. (1996) A highly efficient cell-free protein synthesis system from Escherichia coli. Eur. J. Biochem. 239, 881–886.

    Article  CAS  PubMed  Google Scholar 

  24. Patnaik, R. and Swartz, J. R. (1998) E. coli-based in vitro transcription/translation: in vivo-specific synthesis rates and high yields in a batch system. BioTechniques 24, 862–868

    CAS  PubMed  Google Scholar 

  25. Kim, D. M. and Swartz, J. R. (2000) Prolonging cell-free protein synthesis by selective reagent additions. Biotech. Prog. 16, 385–390

    Article  CAS  Google Scholar 

  26. Kim, R. G. and Choi, C. Y. (2001) Expression-independent consumption of substrates in cell-free expression system from Escherichia coli. J. Biotechnol. 84, 27–32

    Article  CAS  PubMed  Google Scholar 

  27. Ryabova, L. A., Vinokurov, L. M., Shekhovtsova, E. A., Alakhov, Y. B., and Spirin, A. S. (1995) Acetyl phosphate as an energy source for bacterial cell-free translation system. Anal. Biochem. 226, 184–186

    Article  CAS  PubMed  Google Scholar 

  28. Kim, D. M. and Swartz, J. R. (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotech. Bioengineering 66, 180–188

    Article  CAS  Google Scholar 

  29. Kim, D.-M. and Swartz, J. R. (2001) Regeneration of adenosine triphosphate from glycolytic intermediates for cell-free protein synthesis. Biotech. Bioeng. 74, 309–316.

    Article  CAS  Google Scholar 

  30. Jewett, M. C. and Swartz, J. R. (2004) Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotech. Bioeng. 86, 19–26.

    Article  CAS  Google Scholar 

  31. Jewett, M. C. and Swartz, J. R. (2004) Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis. Biotechnol. Prog. 20, 102–109.

    Article  CAS  PubMed  Google Scholar 

  32. Spirin, A. S. (1992) Cell-free protein synthesis bioreactor, in Frontiers in Bioprocessing II, (Todd, P., Sikdar, S. K., and Beer, M., ed.), American Chemical Society, Washington, DC, pp. 31–43.

    Google Scholar 

  33. Nakano, H., Shinbata, T., Okumura, R., Sekiguchi, S., Fujishiro, M., and Yamane, T. (1999) Efficient coupled transcription/translation for PCR template by a hollow fiber membrane bioreactor. Biotechnol. Bioeng. 64, 194–199.

    Article  CAS  PubMed  Google Scholar 

  34. Buchberger, B., Mutter, W., and Röder, A. (2002) Matrix reactor: a new scalable principle for cell-free protein expression, in Cell-Free Translation Systems, (Spirin, A. S., ed.), Springer, Germany, pp. 121–128.

    Google Scholar 

  35. Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., et al. (2002) A bilayer cell-free protein synthesis system for high-throughout screening of gene products. FEBS Lett. 514, 102–105.

    Article  CAS  PubMed  Google Scholar 

  36. Ugarov, V. I., Morozov, I. Yu., Jung, G. V., Chetverin, A. B., and Spirin, A. S. (1994) Expression and stability of recombinant RQ-mRNA in cell-free translation systems. FEBS Lett. 341, 131–134.

    Article  CAS  PubMed  Google Scholar 

  37. Biryukov, S. V., Simonenko, P. N., Shirokov, V. A., and Spirin, A.S. (2004) Method for synthesis of polypeptides in cell-free systems. United States Patent no. 6,783,957.

    Google Scholar 

  38. Kozak, M. (1989) Context effects and inefficient initiation at the non-AUG codons in eucaryotic cell-free translation systems. Mol. Cell. Biol. 9, 5073–5080.

    CAS  PubMed  Google Scholar 

  39. Florentz, C. and Giegé, R. (1995) tRNA-like structures in plant viral RNAs, in tRNA: Structure, Biosynthesis, and Function, (Söll, D. and RajBhandary, U. L., eds.), ASM Press, Washington, pp. 141–163.

    Google Scholar 

  40. Danthinne, X., Seurinck, J., Meulewaeter, F., van Montagu, M., and Cornelissen, M. (1993) The 3′ untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro. Mol. Cell. Biol. 13, 3340–3349.

    CAS  PubMed  Google Scholar 

  41. Timmer, R. T., Benkowski, L. A., Schodin, D., et al. (1993) The 5′ and 3′ untranslated regions of satellite tobacco necrosis virus RNA affect translational efficiency and dependence on a 5′ cap structure. J. Biol. Chem. 268, 9504–9510.

    CAS  PubMed  Google Scholar 

  42. Wang, S. and Miller, W. A. (1995) A sequence located 4.5 to 5 kilobases from the 5′-end of the barley yellow dwarf virus (PAV) genome strongly stimulates translation of uncapped mRNA. J. Biol. Chem. 270, 13,446–13,452.

    Article  CAS  PubMed  Google Scholar 

  43. Gallie, D. R. and Walbot, V. (1990) RNA preudoknot domain of tobacco mosaic virus can functionally substitute for a poly(A) tail in plant and animal cells. Genes Dev. 4, 1149–1157.

    Article  CAS  PubMed  Google Scholar 

  44. Gallie, D. R., Feder, J. N., Schimke, R. T., and Walbot, V. (1991) Functional analysis of the tobacco mosaic virus tRNA-like structure in cytoplasmic gene regulation. Nucleic Acids Res. 19, 5031–5036.

    Article  CAS  PubMed  Google Scholar 

  45. Ryabova, L. A., Torgashov, A. F., Kurnasov, O. V., Bubunenko, M. G., and Spirin, A. S. (1993) The 3′-terminal region of alfalfa mosaic virus RNA4 facilitates the RNA entry into translation in a cell-free system. FEBS Lett. 326, 264–266.

    Article  CAS  PubMed  Google Scholar 

  46. Zeyenko, V. V., Ryabova, L. A., Gallie, D. R., and Spirin, A. S. (1994) Enhancing effect of the 3′-untranslated region of tobacco mosaic virus RNA on protein synthesis in vitro. FEBS Lett. 354, 271–273.

    Article  CAS  PubMed  Google Scholar 

  47. Kawarasaki, Y., Kasahara, S., Kodera, N., et al. (2000) A trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression. Biothechnol. Prog. 16, 517–521.

    Article  CAS  Google Scholar 

  48. Akbergenov, R. Zh., Zhanybekova, S. Sh., Kryldakov, R. V., et al. (2004) ARC-1, a sequence element complementary to an internal 18S rRNA segment, enhances translation efficiency in plants when present in the leader or intercistronic region of mRNAs. Nucleic Acids Res. 32, 239–247

    Article  CAS  PubMed  Google Scholar 

  49. Shaloiko, L. A., Granovsky, I. E., Ivashina, T. V., Ksenzenko, V. N., Shirokov, V. A., and Spirin, A. S. (2004) Effective non-viral leader for cap-independent translation in a eukaryotic cell-free system. Biotechnol. Bioeng. 88, 730–739.

    Article  CAS  PubMed  Google Scholar 

  50. Kolb, V. A., Makeyev, E. V., and Spirin, A. S. (2000) Co-translational folding of an eukaryotic multidomain protein in a prokaryotic translation system. J. Biol. Chem. 275, 16,597–16,601.

    Article  CAS  PubMed  Google Scholar 

  51. Kommer, A., Dashkova, I. G., Yesipov, R. S., Miroshnikov, A. I., and Spirin, A. S. (2005) Synthesis of functionally active human proinsulin in a cell-free translation system. Dokl. Akad. Nauk. 401, 1–5 (Dokl. Biochem. Biophys. 401,154–158).

    Google Scholar 

  52. Nakano, H., Tanaka, T., Kawarasaki, Y., and Yamane, T. (1994) An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. Biosci. Biotechnol. Biochem. 58, 631–634.

    Article  CAS  PubMed  Google Scholar 

  53. Yamane, T., Kawarasaki, Y., and Nakano, H. (1995) In vitro protein biosynthesis using ribosome and foreign mRNA: an approach to construct a protein biosynthesizer. Ann. NY Acad. Sci. 750, 146–157.

    Article  CAS  PubMed  Google Scholar 

  54. Komar, A. A., Kommer, A., Krasheninnikov, I. A., and Spirin, A. S. (1993) Cotranslational heme binding to nascent globin chains. FEBS Lett. 326, 261–263.

    Article  CAS  PubMed  Google Scholar 

  55. Komar, A. A., Kommer, A., Krasheninnikov, I. A., and Spirin, A. S. (1997) Cotranslational folding of globin. J. Biol. Chem. 272, 10,646–10,651.

    Article  CAS  PubMed  Google Scholar 

  56. Mouat, M. F. (2000) Dihydrofolate influences the activity of Escherichia coli dihydrofolate reductase synthesized de novo. Int. J. Biochem. Cell Biol. 32, 327–337.

    Article  CAS  PubMed  Google Scholar 

  57. Knapp, K. G. and Swartz, J. R. (2004) Cell-free production of active E. coli thioredoxin reductase and glutathione reductase. FEBS Lett. 559, 66–70.

    Article  CAS  PubMed  Google Scholar 

  58. Miyazaki-Imamura, C., Oohira, K., Kitagawa, R., Nakano, H., Yamane, T., and Takahashi, H. (2003) Improvement of H2O2 stability of manganese peroxidase by combinatorial mutagenesis and high throughput screening using in vitro expression with protein disulfide isomerase. Protein Eng. 16, 423–428.

    Article  CAS  PubMed  Google Scholar 

  59. Agashe, V. R., Guha, S., Chang, H.-C., et al. (2004) Function of trigger factor and DnaK in multidomain protein folding: increase in yield at the expense of folding speed. Cell 117, 199–209.

    Article  CAS  PubMed  Google Scholar 

  60. Shimizu, Y., Inoue, A., Tomary, Y., et al. (2001) Cell-free translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755.

    Article  CAS  PubMed  Google Scholar 

  61. Svetlov, M. S., Kommer, A., Kolb, V. A., and Spirin, A. S. (2006) Effective cotranslational folding of firefly luciferase without chaperones of Hsp70 family. Prot. Sci. 15, 242–247.

    Article  CAS  Google Scholar 

  62. Ryabova, L. A., Desplancq, D., Spirin, A. S., and Pluckthun, A. (1997) Functional antibody production using cell-free translation: effects of protein disulfide isomerase and chaperones. Nat. Biotechnol. 15, 79–84.

    Article  CAS  PubMed  Google Scholar 

  63. Kudlicki, W., Mouat, M., Walterscheid, J. P., Kramer, G., and Hardesty, B. (1994) Development of a chaperone-deficient system by fractionation of a prokaryotic coupled transcription/translation system. Anal. Biochem. 217, 12–19.

    Article  CAS  PubMed  Google Scholar 

  64. Spirin, A. S., ed. (2002) Cell-Free Translation Systems. Springer-Verlag, Heidelberg, Berlin, New York.

    Google Scholar 

  65. Swartz, J. R., ed. (2003) Cell-Free Protein Expression. Springer-Verlag, Berlin, Heidelberg, New York.

    Google Scholar 

  66. Jiang, X., Ookubo, Y., Fujii, I., Nakano, H., and Yamane, T. (2002) Expression of Fab fragment of catalytic antibody 6D9 in an Escherichia coli in vitro coupled transcription/translation system. FEBS Lett. 514, 290–294.

    Article  CAS  PubMed  Google Scholar 

  67. Yin, G. and Swartz, J. R. (2004) Enhancing multiple disulfide bonded protein folding in a cell-free system. Biotechnol. Bioeng. 86, 188–195.

    Article  CAS  PubMed  Google Scholar 

  68. Yang, J., Kanter, G., Voloshin, A., Levy, R., and Swartz, J. R. (2004) Expression of active murine granulocyte-macrophage colony-stimulating factor in an Escherichia coli cell-free system. Biotechnol. Prog. 20, 1689–1696.

    Article  CAS  PubMed  Google Scholar 

  69. Kim, D.-M. and Swartz, J. R. (2004) Efficient production of a bioactive, multiple disulfide-bonded protein using modified extracts of Escherichia coli. Biotechnol. Bioeng. 85, 122–129.

    Article  CAS  PubMed  Google Scholar 

  70. Steigerwald, R., Nemetz, C., Walckhoff, B., and Emrich, T. (2002) Cell-free expression of a 127 kDa protein: the catalytic subunit of human telomerase, in Cell-Free Translation Systems, (Spirin, A. S., ed.), Springer-Verlag, Heidelberg, Berlin, New York, pp. 165–173.

    Google Scholar 

  71. Nemetz, C., Wessner, S., Krupka, S., Watzele, M., and Mutter, W. (2002) Cell-free expression of soluble human erythropoietin, in Cell-Free Translation Systems, (Spirin, A. S., ed.), Springer-Verlag, Heidelberg, Berlin, New York, pp. 157–163.

    Google Scholar 

  72. Maurer, P., Moratzky, A., Fecher-Trost, C., et al. (2003) Cell-free synthesis of membrane proteins on a preparative scale, in Cell-Free Protein Expression, (Swartz, J. R., ed.), Springer-Verlag, Berlin, Heidelberg, New York, pp. 133–139.

    Google Scholar 

  73. Busso, D., Kim, R., and Kim, S. H. (2003) Expression of soluble recombinant proteins in a cell-free system using a 96-well format. J. Biochem. Biophys. Methods 55, 233–240.

    Article  CAS  PubMed  Google Scholar 

  74. Busso, D., Kim, R., and Kim, S. H. (2004) Using an Escherichia coli cell-free extract to screen for soluble expression of recombinant proteins. J. Struct. Funct. Genomics 5, 69–74.

    Article  CAS  PubMed  Google Scholar 

  75. Klammt, C., Löhr, F., Schafer, B., et al. (2004) High level cell-free expression and specific labeling of integral membrane proteins. Eur. J. Biochem. 271, 568–580.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

    Vladimir A. Shirokov, Aigar Kommer, Vyacheslav A. Kolb & Alexander S. Spirin

Authors
  1. Vladimir A. Shirokov
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Aigar Kommer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Vyacheslav A. Kolb
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alexander S. Spirin
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Biochemistry and Molecular Biology Unit, Novartis Vaccines, Siena, Italy

    Guido Grandi

Rights and permissions

Reprints and permissions

Copyright information

© 2007 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Shirokov, V.A., Kommer, A., Kolb, V.A., Spirin, A.S. (2007). Continuous-Exchange Protein-Synthesizing Systems. In: Grandi, G. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology™, vol 375. Humana Press. https://doi.org/10.1007/978-1-59745-388-2_2

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-388-2_2

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-558-3

  • Online ISBN: 978-1-59745-388-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation