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Biochemistry (Moscow)

, Volume 70, Issue 7, pp 777–781 | Cite as

Recombinant Human Ribosomal Protein S16: Expression, Purification, Refolding, and Structural Stability

  • N. M. Parakhnevitch
  • A. A. Malygin
  • G. G. KarpovaEmail author
Article

Abstract

The cDNA of human ribosomal protein S16 was cloned into the expression vector pET-15b. Large-scale production of the recombinant protein was carried out in E. coli cells and highly purified protein was isolated. A method for refolding the protein from inclusion bodies was optimized. The secondary structure content of the refolded protein was analyzed by CD spectroscopy. It was found that 21 ± 4% of the amino acid sequence of the protein forms α-helices and 24 ± 3% is in β-strands. The protein structure stability was studied at various pH values and urea concentrations. The protein is quickly denatured at pH above 8.0, whereas increasing of urea concentration causes slow unfolding of the protein.

Key words

human ribosomal protein S16 expression refolding 

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REFERENCES

  1. 1.
    Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Jr., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature, 407, 327–339.CrossRefPubMedGoogle Scholar
  2. 2.
    Schluenzen, F., Tocilj, A., Zarivach, R., Harms, J., Gluehmann, M., Janell, D., Bashan, A., Bartels, H., Agmon, I., Franceschi, F., and Yonath, A. (2000) Cell, 102, 615–623.CrossRefPubMedGoogle Scholar
  3. 3.
    Cate, J. H., Yusupov, M. M., Yusupova, G. Z., Earnest, T. N., and Noller, H. F. (1999) Science, 285, 2095–2104.CrossRefPubMedGoogle Scholar
  4. 4.
    Morgan, D. G., Menetreit, J.-F., Neuhof, A., Rapoport, T. A., and Akey, Ch. W. (2002) J. Mol. Biol., 324, 874–886.CrossRefGoogle Scholar
  5. 5.
    Dube, P., Bacher, G., Stark, H., Mueller, F., Zemlin, F., van Heel, M., and Brimacombe, R. (1998) J. Mol. Biol., 279, 403–421.CrossRefPubMedGoogle Scholar
  6. 6.
    Wool, I. G. (1996) Trends Biochem. Sci., 21, 164–165.CrossRefPubMedGoogle Scholar
  7. 7.
    Vilardell, J., Chartrand, P., Singer, R. H., and Warner, J. R. (2000) RNA, 6, 1773–1780.CrossRefPubMedGoogle Scholar
  8. 8.
    Mazumder, B., Sampath, P., Seshadri, V., Maitra, R. K., DiCorleto, P. E., and Fox, P. L. (2003) Cell, 115, 187–198.CrossRefPubMedGoogle Scholar
  9. 9.
    Vladimirov, S. N., Ivanov, A. V., Karpova, G. G., Musolyamov, A. K., Egorov, T. A., Thiede, B., Wittmann-Liebold, B., and Otto, A. (1996) Eur. J. Biochem., 239, 144–149.CrossRefPubMedGoogle Scholar
  10. 10.
    Wool, I. G., Chan, Y.-L., and Glueck, A. (1996) in Translational Control (Hershey, J. W. B., Matthews, M. B., and Sonenberg, N., eds.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 685–732.Google Scholar
  11. 11.
    Malygin, A. A., Shaulo, D. D., and Karpova, G. G. (2000) Biochim. Biophys. Acta, 1494, 213–216.PubMedGoogle Scholar
  12. 12.
    Malygin, A., Baranovskaya, O., Ivanov, A., and Karpova, G. (2003) Protein Exp. Purif., 28, 57–62.CrossRefGoogle Scholar
  13. 13.
    Tsumoto, K., Ejima, D., Kumagai, I., and Arakawa, T. (2003) Protein Exp. Purif., 28, 1–8.CrossRefGoogle Scholar
  14. 14.
    Bradford, M. M. (1976) Analyt. Biochem., 72, 248–254.PubMedGoogle Scholar
  15. 15.
    Sreerama, N., Venyaminov, S. Yu., and Woody, R. W. (1999) Protein Sci., 8, 370–380.PubMedGoogle Scholar
  16. 16.
    Garnier, J., Gibrat, J.-F., and Robson, B. (1996) Meth. Enzymol., 266, 540–553.PubMedGoogle Scholar
  17. 17.
    Jones, D. T. (1999) J. Mol. Biol., 292, 195–202.CrossRefPubMedGoogle Scholar
  18. 18.
    Pollastri, G., Przybylski, D., Rost, B., and Baldi, P. (2002) Proteins, 47, 228–235.CrossRefPubMedGoogle Scholar
  19. 19.
    Brodersen, D. E., Clemons, W. M., Jr., Carter, A. P., Wimberly, B. T., and Ramakrishnan, V. (2002) J. Mol. Biol., 316, 725–768.CrossRefPubMedGoogle Scholar

Copyright information

© MAIK “Nauka/Interperiodica” 2005

Authors and Affiliations

  • N. M. Parakhnevitch
    • 1
  • A. A. Malygin
    • 1
  • G. G. Karpova
    • 1
    Email author
  1. 1.Institute of Chemical Biology and Fundamental MedicineSiberian Branch of the Russian Academy of SciencesNovosibirskRussia

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