Abstract
Cell-free biology exploits and studies complex biological processes in a controlled environment without intact cells. One model system is prokaryotic cell-free protein synthesis. This technology offers an attractive and convenient approach to produce properly folded recombinant DNA (rDNA) proteins on a laboratory scale, screen PCR fragment libraries in a high-throughput format, express pharmaceutical proteins, incorporate labeled or unnatural amino acids into proteins, and activate microbial physiology to allow for investigation of biological systems. We describe the preparation of materials necessary for the expression, quantification, and purification of rDNA proteins from active Escherichia coli extracts.
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
Jewett, M. C., Voloshin, A., and Swartz, J. R. (2002) Prokaryotic systems for in vitro expression, in Gene Cloning and Expression Technologies (Weiner, M. P., and Lu, Q., eds.), Eaton Publishing, Westborough, MA, pp. 391–411.
Kim, D. M., and Swartz, J. R. (2001) Regeneration of ATP from glycolytic intermediates for cell-free protein synthesis. Biotechnol. Bioeng. 74, 309–316.
Jermutus, L., Ryabova, L. A., and Pluckthun, A. (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr. Opin. Biotechnol. 9, 534–548.
Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., et al. (1999) Cell-free production and stable-isotope labeling of milligram quantities of proteins. FEBS Lett. 442, 15–19.
Kim, R. G. and Choi, C. Y. (2000) Expression-independent consumption of substrates in cell-free expression system from Escherichia coli. J. Biotechnol. 84, 27–32.
Kim, D. M. and Swartz, J. R. (1999) Prolonging cell-free protein synthesis with a novel ATP regeneration system. Biotechnol. Bioeng. 66, 180–188.
Nakano, H. and Yamane, T. (1998) Cell-free protein synthesis systems. Biotechnol. Adv. 16, 367–384.
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.
Stiege, W. and Erdmann, V. A. (1995) The potentials of the in vitro protein biosynthesis system. J. Biotechnol. 41, 81–90.
Yokoyama, S., Matsuo, Y., Hirota, H., Kigawa, T., Shirouzu, M., Kuroda, Y., et al. (2000) Structural genomics projects in Japan. Pro. Biophys. Mol. Biol. 72, 363–376.
Alimov, A. P., Khmelnitsky, A. Y., Simonenko, P. N., Spirin, A. S., and Chetverin, A. B. (2000) Cell-free synthesis and affinity isolation of proteins on a nanomole scale. BioTechniques 28, 338–344.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, A Laboratory Manual, 2 ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Davanloo, P., Rosenberg, A. H., Dunn, J. J., and Studier, F. W. (1984) Cloning and expression of the gene for bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 81, 2035–2039.
Pratt, J. M. (1984) Coupled transcription-translation in prokaryotic cell-free systems, in Transcription and Translation: A Practical Approach (Hames, B. D. and Higgins, S. J., eds.), IRL Press, New York, NY, pp. 179–209.
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.
Shaw, W. V. (1975) Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 43, 737–755.
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. In press.
Muller, D. K., Martin, C. T., and Coleman, J. E. (1988) Processivity of proteolytically modified forms of T7 RNA polymerase. Biochemistry 27, 5763–5771.
Michel-Reydellet, N., Calhoun, K. A., and Swartz, J. R. (2004) Amino acid stabilization for cell-free protein synthesis by modification of the E. coli genome. Metabolic Engineering. In press.
Schindler, P. T., Baumann, S., Reuss, M., and Siemann, M. (2000) In vitro transcription translation: effects of modification in lysate preparation on protein composition and biosynthesis activity. Electrophoresis 21, 2606–2609.
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.
Kim, D. M. and Swartz, J. R. (2000) Prolonging cell free protein synthesis by selective reagent additions. Biotechnol. Prog. 16, 385–390.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Swartz, J.R., Jewett, M.C., Woodrow, K.A. (2004). Cell-Free Protein Synthesis With Prokaryotic Combined Transcription-Translation. In: Balbás, P., Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 267. Humana Press. https://doi.org/10.1385/1-59259-774-2:169
Download citation
DOI: https://doi.org/10.1385/1-59259-774-2:169
Publisher Name: Humana Press
Print ISBN: 978-1-58829-262-9
Online ISBN: 978-1-59259-774-1
eBook Packages: Springer Protocols