Springer Protocols: Abstract: Continuous-Exchange Protein-Synthesizing Systems

Continuous-Exchange Protein-Synthesizing Systems

By: Vladimir A. Shirokov², Aigar Kommer², Vyacheslav A. Kolb², Alexander S. Spirin²

Abstract

Full Text

Download PDF (286K)

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.

Affiliation(s): (2) Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, Russia

Book Title: In Vitro Transcription and Translation Protocols

Series: Methods in Molecular Biology | Volume: 375 | Pub. Date: May-03-2007 | Page Range: 19-55 | DOI: 10.1007/978-1-59745-388-2_2

Subject: Genetics/Genomics

Key Words: Continuous-exchange cell-free (CECF) system - E. coli extract - wheat germ extract - translation reaction mixture - combined transcription-translation - 5′-untranslated region (5′-UTR) - 3′-untranslated region (3′-UTR) - protein folding - molecular chaperones - nonionic detergents

References

Download 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.

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.

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

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.

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.

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.

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.

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

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.

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

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.

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.

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.

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.

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.

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

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

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

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

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

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

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.

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.

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

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

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

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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

60a.	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.

61.	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.

62.	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.

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

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

65.	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.

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

67.	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.

68.	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.

69.	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.

70.	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.

71.	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.

72.	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.

73.	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.

74.	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.

Comments (Loading...)

Post comment/View all comments