Abstract
Multi-wavelength anomalous diffraction phasing is especially useful for high-throughput structure determinations. Selenomethionine substituted proteins are commonly used for this purpose. However, the cytotoxicity of selenomethionine drastically reduces the efficiency of its incorporation in in vivo expression systems. In the present study, an improved E. coli cell-free protein synthesis system was used to incorporate selenomethionine into a protein, so that highly efficient incorporation could be achieved. A milligram quantity of selenomethionine-containing Ras was obtained using the cell-free system with dialysis. The mass spectrometry analysis showed that more than 95% of the methionine residues were substituted with selenomethionine. The crystal of this protein grew under the same conditions and had the same unit cell constants as those of the native Ras protein. The three-dimensional structure of this protein, determined by multi-wavelength anomalous diffraction phasing, was almost the same as that of the Ras protein prepared by in vivo expression. Therefore, the cell-free synthesis system could become a powerful protein expression method for high-throughput structure determinations by X-ray crystallography.
Similar content being viewed by others
References
Hendrickson, W.A. (1991) Science 254, 51–58.
Hendrickson, W.A., Horton, J.R. and LeMaster, D.M. (1990) EMBO J. 9, 1665–1672.
O'Gara, M., Adams, G.M., Gong, W., Kobayashi, R., Blumenthal, R.M. and Cheng, X. (1997) Eur. J. Biochem. 247, 1009–1018.
Kigawa, T., Muto, Y. and Yokoyama, S. (1995) J. Biomol. NMR 6, 129–134.
Yabuki, T., Kigawa, T., Dohmae, N., Takio, K., Terada, T., Ito, Y., Laue, E.D., Cooper, J.A., Kainosho, M. and Yokoyama, S. (1998) J. Biomol. NMR 11, 295–306.
Kigawa, T., Yabuki, T., Yoshida, Y., Tsutsui, M., Ito, Y., Shibata, T. and Yokoyama, S. (1999) FEBS Lett. 442, 15–19.
Kim, D.-M., Kigawa, T., Choi, C.-Y. and Yokoyama, S. (1996) Eur. J. Biochem. 239, 881–886.
Madin, K., Sawasaki, T., Ogasawara, T. and Endo, Y. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 559–564.
Davis, J., Thompson, D. and Beckler, G.S. (1996) Promega Notes Magazine 56, 14–18.
Kim, D.M. and Choi, C.Y. (1996) Biotechnol. Prog. 12, 645-649.
Yokoyama, S., Hirota, H., Kigawa, T., Yabuki, T., Shirouzu, M., Terada, T., Ito, Y., Matsuo, Y., Kuroda, Y., Nishimura, Y., Kyogoku, Y., Miki, K., Masui, R. and Kuramitsu, S. (2000) Nat. Struct. Biol. 7 Suppl., 943–945.
de Vos, A.M., Tong, L., Milburn, M.V., Matias, P.M., Jancarik, J., Noguchi, S., Nishimura, S., Miura, K., Ohtsuka, E. and Kim, S.H. (1988) Science 239, 888–893.
Pratt, J.M. (1984) In Transcription and translation (Eds., Hames, B.D. and Higgins, S.J.), IRL Press, Oxford, Washington DC, pp. 179–209.
Zawadzki, V. and Gross, H.J. (1991) Nucleic Acids Res. 19, 1948.
Ha, J.-M., Ito, Y., Kawai, G., Miyazawa, T., Miura, K., Ohtsuka, E., Noguchi, S., Nishimura, S. and Yokoyama, S. (1989) Biochemistry 28, 8411–8416.
Yamasaki, K., Kawai, G., Ito, Y., Muto, Y., Fujita, J., Miyazawa, T., Nishimura, S. and Yokoyama, S. (1989) Biochem. Biophys. Res. Commun. 162, 1054–1062.
Akashi, S., Shirouzu, M., Yokoyama, S. and Takio, K. (1996) J. Mass Spectrom. Soc. Jpn. 44, 269–277.
Otwinowski, Z. and Minor, W. (1997) Macromol. Crystallogr. A276, 307–326.
Knight, S.D. (2000) Acta Crystallogr. D56, 42–47.
de la Fortelle, E. and Bricogne, G. (1997) Methods Enzymol. 276, 472–494.
Hauptman, H.A., Xu, H., Weeks, C.M. and Miller, R. (1999) Acta Crystallogr. 55, 891–900.
Collaborative Computational Project Number 4 (1994) Acta Crystallogr. D50, 760–763.
Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. (1991) Acta Crystallogr. A47, 110–119.
Brünger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T. and Warren, G.L. (1998) Acta Crystallogr. D54, 905–921.
Kraulis, P.J., Domaille, P.J., Campbell-Burk, S.L., Van Aken, T. and Laue, E.D. (1994) Biochemistry, 33, 3515–3531.
Ito, Y., Yamasaki, K., Iwahara, J., Terada, T., Kamiya, A., Shirouzu, M., Muto, Y., Kawai, G., Yokoyama, S., Laue, E.D., Wälchli, M., Shibata, T., Nishimura, S. and Miyazawa, T. (1997) Biochemistry 36, 9109–9119.
Tsalkova, T., Zardeneta, G., Kudlicki, W., Kramer, G., Horowitz, P.M. and Hardesty, B. (1993) Biochemistry 32, 3377–3380.
Ryabova, L.A., Desplancq, D., Spirin, A.S. and Pluckthun, A. (1997) Nat. Biotechnol. 15, 79–84.
Cayanis, E., Bayley, H. and Edelman, I.S. (1990) J. Biol. Chem. 265, 10829–10835.
Markoff, L. (1989) J. Virol. 63, 3345–3352.
Martemyanov, K.A., Shirokov, V.A., Kurnasov, O.V., Gudkov, A.T. and Spirin, A.S. (2001) Protein Expr. Purif. 21, 456–461.
Koradi, R., Billeter, M. and Wüthrich, K. (1996) J. Mol. Graph. 14, 51–55.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kigawa, T., Yamaguchi-Nunokawa, E., Kodama, K. et al. Selenomethionine incorporation into a protein by cell-free synthesis. J Struct Func Genom 2, 29–35 (2002). https://doi.org/10.1023/A:1013203532303
Issue Date:
DOI: https://doi.org/10.1023/A:1013203532303