Journal of Biomolecular NMR

, Volume 30, Issue 3, pp 311–325 | Cite as

Efficient production of isotopically labeled proteins by cell-free synthesis: A practical protocol

  • Takuya Torizawa
  • Masato Shimizu
  • Masato Taoka
  • Hiroshi Miyano
  • Masatsune KainoshoEmail author


We provide detailed descriptions of our refined protocols for the cell-free production of labeled protein samples for NMR spectroscopy. These methods are efficient and overcome two critical problems associated with the use of conventional Escherichia coli extract systems. Endogenous amino acids normally present in E. coli S30 extracts dilute the added labeled amino acids and degrade the quality of NMR spectra of the target protein. This problem was solved by altering the protocol used in preparing the S30 extract so as to minimize the content of endogenous amino acids. The second problem encountered in conventional E. coli cell-free protein production is non-uniformity in the N-terminus of the target protein, which can complicate the NMR spectra. This problem was solved by adding a DNA sequence to the construct that codes for a cleavable N-terminal peptide tag. Addition of the tag serves to increase the yield of the protein as well as to ensure a homogeneous protein product following tag cleavage. We illustrate the method by describing its stepwise application to the production of calmodulin samples with different stable isotope labeling patterns for NMR analysis.


cell-free extract from E. coli cell-free protein synthesis NMR assignments protocol optimization stable isotope labeling 


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  1. Betton, J.M. 2003Curr. Protein Pept. Sci.47380Google Scholar
  2. Bradford, M.M. 1976Anal. Biochem.72248254CrossRefPubMedGoogle Scholar
  3. Chrunyk, B.A., Evans, J., Lillquist, J., Young, P., Wetzel, R. 1993J. Biol. Chem.2681805318061Google Scholar
  4. Clemens, M.J., Prujin, G.J. 1999Protein Synthesis in Eukaryotic Cell-Free SystemsOxford University PressNew York129165Google Scholar
  5. Clore, G.M., Gronenborn, A.M. 1994Meth. Enzymol.239349363Google Scholar
  6. Davanloo, P., Rosenberg, A.H., Dunn, J.J., Studier, F.W. 1984Proc. Natl. Acad. Sci. USA8120352039Google Scholar
  7. Goff, S.A., Goldberg, A.L. 1987J. Biol. Chem.26245084515Google Scholar
  8. Grodberg, J., Dunn, J.J. 1988J. Bacteriol.17012451253Google Scholar
  9. Guignard, L., Ozawa, K., Pursglove, S.E., Otting, G., Dixon, N.E. 2002FEBS Lett.524159162Google Scholar
  10. Henrich, B., Lubitz, W., Plapp, R. 1982Mol. Gen. Genet.185493497Google Scholar
  11. Ikura, M., Kay, L.E., Bax, A. 1990aBiochemistry2946594667Google Scholar
  12. Ikura, M., Marion, D., Kay, L.E., Shih, H., Krinks, M., Klee, C.B., Bax, A. 1990bBiochem. Pharmacol.40153160Google Scholar
  13. Kariya, E., Ohki, S., Hayano, T., Kainosho, M. 2000J. Biomol. NMR.187576Google Scholar
  14. Kelly, A.E., Ou, H.D., Withers, R., Dötsch, V. 2002J. Am. Chem. Soc.1241201312019Google Scholar
  15. Kigawa, T., Muto, Y., Yokoyama, S. 1995J. Biomol. NMR6129134Google Scholar
  16. Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T., Yokoyama, S. 1999FEBS Lett.4421519Google Scholar
  17. Kim, D.M., Swartz, J.R. 2000Biotechnol. Prog.16385390Google Scholar
  18. Kim, D.M., Kigawa, T., Choi, C.Y., Yokoyama, S. 1996Eur. J. Biochem.239881886Google Scholar
  19. Klammt, C., Lohr, F., Schafer, B., Haase, W., Dötsch, V., Rüterjans, H., Glaubitz, C., Bernhard, F. 2004Eur. J. Biochem.271568580Google Scholar
  20. Kramer, G., Kudlicki, W., Hardesty, B. 1999Cell-free Coupled Transcription–Translation Systems from Escherichia coliOxford University PressNew York129165Google Scholar
  21. Madin, K., Sawasaki, T., Ogasawara, T., Endo, Y. 2000Proc. Natl. Acad. Sci. USA97559564Google Scholar
  22. Markley, J.L., Bax, A., Arata, Y., Hilbers, C.W., Kaptein, R., Sykes, B.D., Wright, P.E., Wüthrich, K. 1998Eur. J. Biochem.256115Google Scholar
  23. Maurizi, M.R. 1987J. Biol. Chem.26226962703Google Scholar
  24. Mori, S., Abeygunawardana, C., Johnson, M.O., Zijl, P.C. 1995J. Magn. Reson. B1089498Google Scholar
  25. Pratt, J.M. 1984Transcription and Translation: A Practical ApproachIRL PressNew York179209Google Scholar
  26. Shi, J., Pelton, J.G., Cho, H.S., Wemmer, D.E. 2004J. Biomol. NMR28235247Google Scholar
  27. Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., Ueda, T. 2001Nat. Biotechnol.19751755Google Scholar
  28. Spirin, A.S., Baranov, V.I., Ryabova, L.A., Ovodov, S.Y., Alakhov, Y.B. 1988Science24211621164Google Scholar
  29. Wishart, D.S., Bigam, C.G., Yao, J., Abildgaard, F., Dyson, H.J., Oldfield, E., Markley, J.L., Sykes, B.D. 1995J. Biomol. NMR6135140Google Scholar
  30. Zawadzki, V., Gross, H.J. 1991Nucl. Acids Res.191948Google Scholar
  31. Zubay, G. 1973Annu. Rev. Genet.7267287Google Scholar
  32. Zwahlen, C., Legault, P., Vincent, S.J.F., Greenblatt, J., Konrat, R., Kay, L.E. 1997J. Am. Chem. Soc.11967116721Google Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Takuya Torizawa
    • 1
  • Masato Shimizu
    • 1
  • Masato Taoka
    • 2
  • Hiroshi Miyano
    • 3
  • Masatsune Kainosho
    • 1
    • 2
    Email author
  1. 1.Crest, JstJapan
  2. 2.Graduate School of ScienceTokyo Metropolitan UniversityHachiojiJapan
  3. 3.Ajinomoto Co., Inc. Institute of Life SciencesKwasakiJapan

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