Selenomethionine Labeling of Recombinant Proteins

  • Anna M. Larsson
  • T. Alwyn Jones
Part of the Methods in Molecular Biology book series (MIMB, volume 389)


Selenomethionine incorporation is a standard method for determining the phases in protein crystallography by single- or multiwavelength anomalous dispersion. Recombinant expression of selenomethionine-containing protein in non-auxotrophic Pichia pastoris strains yield an incorporation of about 50%. The expression of a mutated variant of Penicillium minioluteum dextranase in P. pastoris is used to illustrate the method utilized to obtain selenomethionyl-substituted protein and to show the phasing power of the acquired anomalous signal. The dextranase structure was solved using the anomalous signal achieved from 50% selenomethionine incorporation.

Key Words

Dextranase MAD multiwavelength anomalous dispersion Pichia pastoris SeMet selenomethionine 


  1. 1.
    Hendrickson, W. A. (1991) Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. Science 254, 51–58.CrossRefPubMedGoogle Scholar
  2. 2.
    Hendrickson, W. A., Horton, J. R., and LeMaster, D. M. (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J. 9, 1665–1672.PubMedGoogle Scholar
  3. 3.
    Van Duyne, G. D., Standaert, R. F., Karplus, P. A., Schreiber, S. L., and Clardy, J. (1993) Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin. J. Mol. Biol. 229, 105–124.CrossRefPubMedGoogle Scholar
  4. 4.
    Doublié, S. (1997) Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530.CrossRefPubMedGoogle Scholar
  5. 5.
    Bushnell, D. A., Cramer, P., and Kornberg, R. D. (2001) Selenomethionine incorporation in Saccharomyces cerevisiae RNA polymerase II. Structure 9, R11–R14.CrossRefPubMedGoogle Scholar
  6. 6.
    Larsson, A. M., Ståhlberg, J., and Jones, T. A. (2002) Preparation and crystallization of selenomethionyl dextranase from Penicillium minioluteum expressed in Pichia pastoris. Acta Cryst, D58, 346–348.Google Scholar
  7. 7.
    Yong, V., González, M. E., Herrera, L., and Delgado, J. (1992) El gen HIS3 complementa una mutación his de Pichia pastoris. Biotecnología Aplicada 9, 55–61.Google Scholar
  8. 8.
    Yong, V., Herrera, L., Margolles, E., et al. (1991) Method for the expression of heterologuos genes in the yeast Pichia pastoris, expression vectors and transformed microorganisms. European Patent Application. Pub. No. 0438 200 A1.Google Scholar
  9. 9.
    McPherson, A. (1982) Preparation and Analysis of Protein Crystals. John Wiley & Sons, New York, NY.Google Scholar
  10. 10.
    Shepherd, L. and Huber, R. E. (1969) Some chemical and biochemical properties of selenomethionine. Can. J. Biochem. 47, 877–881.CrossRefPubMedGoogle Scholar
  11. 11.
    Smith, J. L. and Thompson, A. (1998) Reactivity of selenomethionine-dents in the magic bullet? Structure 6, 815–819.CrossRefPubMedGoogle Scholar
  12. 12.
    Thomazeau, K., Curien, G., Thompson, A., Dumas, R., and Biou, V. (2001) MAD on threonine synthase: the phasing power of oxidized selenomethionine. Acta Cryst. D57, 1337–1340.Google Scholar
  13. 13.
    Sharff, A. J., Koronakis, E., Luisi, B., and Koronakis, V. (2000) Oxidation of selenomethionine: some MADness in the method! Acta Cryst. D56, 785–788.Google Scholar
  14. 14.
    Ravelli, R. B. and McSweeney, S. M. (2000) The “fingerprint” that X-rays can leave on structures. Structure Fold Des. 8, 315–328.CrossRefPubMedGoogle Scholar
  15. 15.
    Larsson, A. M., Andersson, R., Ståhlberg, J., Kenne, L., and Jones, T. A. (2003) Dextranase from Penicillium minioluteum: reaction course, crystal structure, and product complex. Structure 11, 1111–1121.CrossRefPubMedGoogle Scholar
  16. 16.
    Sheldrick, G. M. (2003) SHELX (
  17. 17.
    Schneider, T. R. and Sheldrick, G. M. (2002) Substructure solution with SHELXD. Acta Cryst. D58, 1772–1779.Google Scholar
  18. 18.
    Bricogne, G., Vonrhein, C., Paciorek, W., et al. (2002) Enhancements in autoSHARP and SHARP, with applications to difficult phasing problems. Acta Cryst. A58(Supplement), C239.Google Scholar
  19. 19.
    Cowtan, K. (1994) An automated procedure for phase improvement by density modification. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography 31, 34–38.Google Scholar
  20. 20.
    Xu, B., Munoz, I. G., Janson, J. C., and Ståhlberg, J. (2002) Crystallization and X-ray analysis of native and selenomethionyl β-mannanase Man5A from blue mussel, Mytilus edulis, expressed in Pichia pastoris. Acta Cryst. D58, 542–545.Google Scholar
  21. 21.
    Jones, T. A., Zou, J.-Y., Cowan, S. W., and Kjeldgaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A47, 110–119.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Anna M. Larsson
    • 1
  • T. Alwyn Jones
    • 1
  1. 1.Department of Cell and Molecular BiologyUniversity of UppsalaUppsalaSweden

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