Abstract
The continuous inflow of consumable substrates and outflow of products tremendously increase the lifetime of cell-free protein synthesizing systems (1). As a consequence, the protein yield of the continuous-flow cell-free (CFCF) translation can be raised up to two orders of magnitude, as compared with classical batch systems. The CFCF protein synthesis technique can be utilized for basic research in the field of protein synthesis and folding as well as for numerous applications in biotechnology.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Spirin, A. S., Baranov, V. I., Ryabova, L. A., Ovodov, S. Yu, and Alakhov, Yu. B (1988) A continuous cell-free translation system capable of producing polypeptides in high yield. Science 242, 1162–l164.
Ryabova, L. A., Ortlepp, S. A., and Baranov, V. I. (1989) Preparative synthesis of globin in a continuous cell-free translation system from rabbit reticulocytes. Nucl. Acids Res. 17, 4412.
Gallie, D. R., Feder, J. N., Schimke, R. T., and Walbot, V. (1991) Post-transcriptional regulation in higher eukaryotes. the role of the reporter gene in controlling expression. Mol. Gen. Genet. 228, 258–264.
Leathers, V., Tanguay, R., Kobayashi, M., and Gallie, D. R. (1993) A phylogenetically conserved sequence within viral 3′ untranslated RNA pseudoknots regulates translation. Mol. Cell Biol. 13, 5331–5347.
Ryabova, L. A., Torgashov, A. F., Kurnasov, O. V., Bubunenko, M. G., and Spirin, A. S. (1993) 3′-untranslated region of alfalfa mosaic virus RNA 4 facilitates the RNA entry into translation in a cell-free system. FEBS Lett. 326, 264–266.
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.
Timmer, R. T., Benkowski, L. A., Schodin, D., Lax, S. R., Metz, A. M., Ravel, J. M., and Browning, K. S. (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.
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.
Katanaev, V. L., Kurnasov, O. V., and Spirin, A. S. (1995) Viral QB RNA as a high expression vector for mRNA translation in a cell-free system. FEBS Lett. 359, 89–92.
Ugarov, V. I., Morozov, I. Yu., Jung, G. Y., Chetverin, A. B., and Spirin A. S. (1994) Expression and stability of recombinant RQ-mRNAs in cell-free translation systems. FEBS Lett. 341, 131–134.
Baranov, V. I., Morozov, I. Yu., Ortlepp, S. A., and Spirin A. S. (1989) Preparative gene expression in a cell-free system. Gene 84, 463–466.
Kigawa, T. and Yokoyama, S. (1991) A continuous cell-free syntheses system for coupled transcription-translation. J. Biochem. (Japan) 110, 166–168.
Spirin, A. S. (1992) Cell-free protein synthesis bioreactor, in Frontiers of Bioprocessing II (Todd, P., Sikdar, S. K., and Bier, M., eds.), American Chemical Society, Washington, DC, pp. 31–43.
Baranov, V. I. and Spirin, A. S. (1993) Gene expression in cell-free system on preparative scale. Meth. in Enzymol. 217, 123–142.
Kudlicki, W., Kramer, G., and Hardesty, B. (1992) High efficiency cell-free synthesis of proteins refinement of the coupled transcription/translation system. Analyt. Biochem. 206, 389–393.
Chevrier-Miller, M., Jacques, N., Raibaud, O., and Dreyfus, M. (1990) Transcription of single-copy hybrid lacZ genes by T7 RNA polymerase in Escherichia coli: mRNA synthesis and degradation can be uncoupled from translation. Nucl. Acids Res. 18, 5787–5792.
Iost, I., Guillerez, J., and Dreyfus, M. (1992) Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo. J. Bacteriol. 174, 619–622.
Morozov, I. Yu., Ugarov, V. I., Chetverin, A. B., and Sperm A. S. (1993) Synergism in replication and translatton of messenger RNA in a cell-free system. Proc. Natl. Acad. Sci. USA 90, 9325–9329.
Ryabova, L., Volianik, E., Kurnasov, O., Spirin, A., Wu, Y., and Kramer, F. R. (1994) Coupled replication-translation of amplifiable messenger RNA. J. Biol. Chem. 269, 1501–1505.
Chetverin, A. B. and Spirin, A. S. (1995) RQ RNA vectors: prospects for cell-free gene amplification, expression and cloning, in Progress in Nucleic. Acid Research and Molecular Biology (Cohn, W. E. and Moldave, K., eds.) Academic, San Diego, CA, pp. 225–270.
Billeter, M. A., Libonati, M., Vinuela, E., and Weissmann, C. (1966) Replication of viral ribonucleic acid. J. Biol. Chem. 241, 4750–4757.
Hotham-Iglewski, B., Phillips, L. A., and Franklin, R. M. (1968) Viral RNA transcription-translation complex in Escherichia coli infected with bacteriophage R17. Nature 219, 700–703.
Chen, H-Z. and Zubay, G. (1983) Prokaryotic coupled transcription-translation. Meth. Enzymol. 101, 674–690.
Gavrilova, L. P., and Sperin, A. S. (1974) “Nonenzymatic” Translation. Meth. Enzymol. 30, 452–470.
Gold, L. M. and Shweiger, M. (1971) Synthesis of bacteriophage-specific enzymes directed by DNA in vitro. Methods Enzymol. 20, 537–542.
Gurevitch, V. V., Pokrovskaya, I. D., Obukhova, T. A., and Zozulia, S. A. (1991) Preparative in vitro mRNA synthesis using SP6 and T7 RNA polymerases. Analyt. Biochem. 195, 207–213.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4d. Nature 227, 680–685.
Baccanari, D. P., Phillips, A., Smith, S., Sinski, D., and Burchall, J. (1975) Purification and properties of Escherichia coli dihydrofolate reductase. Biochemistry 14, 5267–5273.
Stanley, W. M. and Wahba, A. J. (1967) Chromatographic purification of ribosomes. Meth. Enzymol. (Nucl. Acids, part A) 12, 524–526.
Ryabova, L. A., Vinokurov, L. M., Shekhovtsova, E. A., Alakhov, Yu. B., and Spirin, A. S. (1995) Acetyl phosphate as an energy donor for bacterial cell-free translation systems. Anal. Biochem, 226, 184–186.
Lindner, P., Guth, B, Wulfing, C., Krebber, C., Steipe, B., Müller, F., and Pluckthun, A. (1992) Purification of native proteins from the cytoplasm and periplasm of Escherichia coli using IMAC and histidine tails a comparison of proteins and protocols. Methods: A Companion to Methods in Enzymology 4, 41–56.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc.
About this protocol
Cite this protocol
Ryabova, L.A., Morozov, l.Y., Spirin, A.S. (1998). Continuous-Flow Cell-Free Translation, Transcription-Translation, and Replication-Translation Systems. In: Martin, R. (eds) Protein Synthesis. Methods in Molecular Biology, vol 77. Springer, Totowa, NJ. https://doi.org/10.1385/0-89603-397-X:179
Download citation
DOI: https://doi.org/10.1385/0-89603-397-X:179
Publisher Name: Springer, Totowa, NJ
Print ISBN: 978-0-89603-397-9
Online ISBN: 978-1-59259-563-1
eBook Packages: Springer Protocols