Biochemistry (Moscow)

, Volume 77, Issue 11, pp 1315–1325 | Cite as

Molecular cloning, isolation, and properties of chaperone Skp from Yersinia pseudotuberculosis

  • E. V. SidorinEmail author
  • N. M. Tishchenko
  • V. A. Khomenko
  • M. P. Isaeva
  • P. S. Dmitrenok
  • N. Yu. Kim
  • G. N. Likhatskaya
  • T. F. Solov’eva


The skp gene of Yersinia pseudotuberculosis was expressed without its signal sequence in Escherichia coli BL21(DE3) cells. The recombinant protein Skp accumulated in soluble form in the cytoplasm of the producer strain. The protein was isolated and characterized: the molecular weight, isoelectric point, N-terminal amino acid sequence (20 amino acid residues), and the content of the secondary structure elements were determined. Using cross-linking stabilization and high-mass MALDI-TOF mass spectrometric analysis, it was found that rSkp forms a stable homotrimer in solution and interacts with human IgG. Three-dimensional models of the Skp trimer and its complexes with Fc- and Fab-fragments of human IgG1 were constructed by computer modeling.

Key words

chaperone Skp Yersinia pseudotuberculosis immunoglobulin G cross-linking stabilization MALDI-TOF mass spectrometry computer modeling protein-protein interactions 





matrix-assisted laser desorption ionization time-of-flight mass spectrometry


recombinant chaper-one Skp


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  1. 1.
    Thome, B., Hoffschulte, H., Schiltz, E., and Muller, M. (1990) FEBS Lett., 269, 113–116.PubMedCrossRefGoogle Scholar
  2. 2.
    Thome, B., and Muller, M. (1991) Mol. Microbiol., 5, 2815–2821.PubMedCrossRefGoogle Scholar
  3. 3.
    Chen, R., and Henning, U. (1996) Mol. Microbiol., 19, 1287–1294.PubMedCrossRefGoogle Scholar
  4. 4.
    De Cock, H., Schafer, U., Potgeter, M., Demel, R., Muller, M., and Tommassen, J. (1999) Eur. J. Biochem., 259, 96–103.PubMedCrossRefGoogle Scholar
  5. 5.
    Schafer, U., Beck, K., and Muller, M. (1999) J. Biol. Chem., 274, 24567–24574.PubMedCrossRefGoogle Scholar
  6. 6.
    Qu, J., Mayer, C., Behrens, S., Holst, O., and Kleinschmidt, J. (2007) J. Mol. Biol., 374, 91–105.PubMedCrossRefGoogle Scholar
  7. 7.
    Schlapschy, M., Dommel, M., Hadian, K., Fogarasi, M., Korndorfer, I., and Skerra, A. (2004) Biol. Chem., 385, 137–143.PubMedCrossRefGoogle Scholar
  8. 8.
    Bulieris, P., Behrens, S., Holst, O., and Kleinschmidt, J. H. (2003) J. Biol. Chem., 278, 9092–9099.PubMedCrossRefGoogle Scholar
  9. 9.
    Geyer, R., Galanos, C., Westphal, O., and Golecki, J. (1979) Eur. J. Biochem., 98, 27–38.PubMedCrossRefGoogle Scholar
  10. 10.
    Holck, A., and Kleppe, K. (1988) Gene, 67, 117–124.PubMedCrossRefGoogle Scholar
  11. 11.
    Holck, A., Lossius, I., Aasland, R., and Kleppe, K. (1987) Biochim. Biophys. Acta, 914, 49–54.PubMedCrossRefGoogle Scholar
  12. 12.
    Holck, A., Lossius, I., Aasland, R., Haarr, L., and Kleppe, K. (1987) Biochim. Biophys. Acta, 908, 188–199.PubMedCrossRefGoogle Scholar
  13. 13.
    Koski, P., Rhen, M., Kantele, J., and Vaara, M. (1989) J. Biol. Chem., 264, 18973–18980.PubMedGoogle Scholar
  14. 14.
    Koski, P., Hirvas, L., and Vaara, M. (1990) Gene, 88, 117–120.PubMedCrossRefGoogle Scholar
  15. 15.
    Shrestha, A., Shi, L., Tanase, S., Tsukamoto, M., Nishino, N., Tokita, K., and Yamamoto, T. (2004) Am. J. Pathol., 164, 763–772.PubMedCrossRefGoogle Scholar
  16. 16.
    Vuorio, R., Hirvas, L., Raybourne, R. B., Yu, D. T. Y., and Vaara, M. (1991) Biochim. Biophys. Acta, 1129, 124–126.PubMedCrossRefGoogle Scholar
  17. 17.
    Bothmann, H., and Pluckthun, A. (1998) Nat. Biotechnol., 16, 376–380.PubMedCrossRefGoogle Scholar
  18. 18.
    Chain, P. S. G., Carniel, E., Larimer, F. W., Lamerdin, J., Stoutland, P. O., Regala, W. M., Georgescu, A. M., Vergez, L. M., Land, M. L., Motin, V. L., Brubaker, R. R., Fowler, J., Hinnebusch, J., Marceau, M., Medigue, C., Simonet, M., Chenal-Francisque, V., Souza, B., Dacheux, D., Elliott, J. M., Derbise, A., Hauser, L. J., and Garcia, E. (2004) Proc. Natl. Acad. Sci. USA, 101, 13826–13831.PubMedCrossRefGoogle Scholar
  19. 19.
    Sidorin, E. V., Ziganshin, R. Kh., Naberezhnyh, G. A., Likhatskaya, G. N., Trifonov, E. V., Anastyuk, S. D., Chernikov, O. V., and Solov’eva, T. F. (2009) Biochemistry (Moscow), 74, 406–415.CrossRefGoogle Scholar
  20. 20.
    Sambrook, J., and Russel, D. W. (2001) in Molecular Cloning, Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory Press, N. Y., pp. 16.33–16.36.Google Scholar
  21. 21.
    Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) Mol. Biol. Evol., 24, 1596–1599.PubMedCrossRefGoogle Scholar
  22. 22.
    Sambrook, J., and Russel, D. W. (2001) in Molecular Cloning, Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory Press, N. Y., p. A2.4.Google Scholar
  23. 23.
    Sambrook, J., and Russel, D. W. (2001) in Molecular Cloning, Laboratory Manual, 3rd Edn., Cold Spring Harbor Laboratory Press, N. Y., p. A1.7.Google Scholar
  24. 24.
    Mikhailov, A. T., and Smirnitskii, V. N. (1991) in Methods of Immunochemical Analysis in Developmental Biology (Engelhardt, N. V., ed.) Nauka, Moscow, pp. 162–171.Google Scholar
  25. 25.
    Laemmli, U. K. (1970) Nature, 227, 680–685.PubMedCrossRefGoogle Scholar
  26. 26.
    Gasparov, V. S., and Degtyar’, V. G. (1994) Biochemistry (Moscow), 59, 563–572.Google Scholar
  27. 27.
    Provencher, S. W., and Glokner, J. (1981) Biochemistry, 20, 33–37.PubMedCrossRefGoogle Scholar
  28. 28.
    Molecular Operating Environment (MOE), 2011.10; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2011.Google Scholar
  29. 29.
    Korndorfer, I. P., Dommel, M. K., and Skerra, A. (2004) Nat. Struct. Mol. Biol., 11, 1015–1120.PubMedCrossRefGoogle Scholar
  30. 30.
    Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) Nucleic Acids Res., 28, 235–242.PubMedCrossRefGoogle Scholar
  31. 31.
    Wang, J., Cieplak, P., and Kollman, P. A. (2001) J. Comput. Chem., 21, 1049–1074.CrossRefGoogle Scholar
  32. 32.
    Bond, S. D., Benedict, J. L., and Laird, B. B. (1999) J. Comput. Phys., 151, 114–134.CrossRefGoogle Scholar
  33. 33.
    Katchalski-Katzir, E., Shariv, I., Eisenstein, M., Friesem, A. A., Aflalo, C., and Vakser, I. A. (1992) Proc. Natl. Acad. Sci. USA, 89, 2195–2199.PubMedCrossRefGoogle Scholar
  34. 34.
    Eppinger, M., Rosovitz, M. J., Fricke, W. F., Rasko, D. A., Kokorina, G., Fayolle, C., Lindler, L. E., Carniel, E., and Ravel, J. (2007) PLoS Genet., 3, e142.PubMedCrossRefGoogle Scholar
  35. 35.
    Sorensen, H. P., and Mortensen, K. K. (2005) Microbial. Cell Factories, 4, 1–8.PubMedCrossRefGoogle Scholar
  36. 36.
    Sreerama, N., and Woody, R. W. (2000) Anal. Biochem., 287, 252–260.PubMedCrossRefGoogle Scholar
  37. 37.
    Venyaminov, S. Yu., and Vassilenko, K. S. (1994) Anal. Biochem., 222, 176–184.PubMedCrossRefGoogle Scholar
  38. 38.
    Walton, T. A., and Sousa, M. C. (2004) Mol. Cell, 15, 367–374.PubMedCrossRefGoogle Scholar
  39. 39.
    Osterman, L. A. (1985) in Chromatography of Proteins and Nucleic Acids (Gorozhanin, P. P., ed.) Nauka, Moscow, pp. 146–152.Google Scholar
  40. 40.
    Farmer, T. B., and Caprioli, R. M. (1998) J. Mass Spectrom., 33, 697–704.PubMedCrossRefGoogle Scholar
  41. 41.
    Sinz, A. (2006) Mass Spectrom. Rev., 25, 663–682.PubMedCrossRefGoogle Scholar
  42. 42.
    Pimenova, T., Pereira, C. P., Schaer, D. J., and Zenobi1, R. (2009) J. Sep. Sci., 32, 1224–1230.PubMedCrossRefGoogle Scholar
  43. 43.
    Fadouloglou, V. E., Kokkinidis, M., and Glykos, N. M. (2008) Anal. Biochem., 373, 404–406.PubMedCrossRefGoogle Scholar
  44. 44.
    Vakser, I. A. (1996) Biopolymers, 39, 455–464.PubMedCrossRefGoogle Scholar
  45. 45.
    Katschke, K. J., Wu, P., Ganesan, R., Kelley, R. F., Mathieu, M. A., Hass, P. E., Murray, J., Kirchhofer, D., Wiesmann, C., and van Lookeren Campagne, M. (2012) J. Biol. Chem., 287, 12886–12892.PubMedCrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2012

Authors and Affiliations

  • E. V. Sidorin
    • 1
    Email author
  • N. M. Tishchenko
    • 1
  • V. A. Khomenko
    • 1
  • M. P. Isaeva
    • 1
  • P. S. Dmitrenok
    • 1
  • N. Yu. Kim
    • 1
  • G. N. Likhatskaya
    • 1
  • T. F. Solov’eva
    • 1
  1. 1.Elyakov Pacific Institute of Bioorganic ChemistryFar Eastern Branch of the Russian Academy of SciencesVladivostokRussia

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