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Russian Journal of Bioorganic Chemistry

, Volume 44, Issue 2, pp 178–187 | Cite as

The Effect of Conditions of the Expression of the Recombinant Outer Membrane Phospholipase А1 from Yersinia pseudotuberculosis on the Structure and Properties of Inclusion Bodies

  • S. I. Bakholdina
  • E. V. Sidorin
  • V. A. Khomenko
  • M. P. Isaeva
  • N. Yu. Kim
  • E. P. Bystritskaya
  • E. A. Pimenova
  • T. F. Solov’eva
Article
  • 18 Downloads

Abstract

The effect of the concentration of an inducer (IPTG) and the time of induction at 37°С on the heterologous synthesis of the mature membrane protein phospholipase А1 (PldA) from Yersinia pseudotuberculosis in the form of inclusion bodies (IBs) and on the physicochemical and structural characteristics of IBs has been studied. The sizes, shape, stability (solubility in urea and detergents, resistance against proteolysis), the secondary structure of the protein of IBs, and the presence of amyloid structures have been determined by electron microscopy, dynamic light scattering, and optical spectroscopy. It was found that IBs have a shape close to spherical and a rough surface and are cleaved by proteinase K. The protein contained in IBs has an ordered secondary structure with a high content of β-structure. As the inducer concentration and the time of expression increase, the conformation of the recombinant protein in IBs undergoes changes, as indicated by an increase in the stability of IBs and a decrease in the enzymatic activity of the protein. When IBs are dissolved in 0.06% SDS and 5 M urea, the recombinant protein retains the secondary structure in a partially modified form, and the addition of a zwitterionic detergent at a micellar concentration does not transform the protein conformation into the native one.

Keywords

Yersinia pseudotuberculosis recombinant protein membrane phospholipase А1 inclusion bodies structural organization 

Abbreviations

IB

inclusion body

DLS

dynamic light scattering

SB3-12

N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate

PBS

phosphate-buffered saline, рН 7.5

IPTG

isopropyl β-D-1-thiogalactopyranoside

LPS

lipopolysaccharides

PldA

detergent-resistant phospholipase A1

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References

  1. 1.
    Baneyx, F., Curr. Opin. Biotechnol., 1999, vol. 10, pp. 411–421.CrossRefPubMedGoogle Scholar
  2. 2.
    Jevsevar, S., Gaberc-Porekar, V., Fonda, I., Podobnik, B., Grdadolnik, J., and Menart, V., Biotechnol. Prog., 2005, vol. 21, pp. 632–639.CrossRefPubMedGoogle Scholar
  3. 3.
    Singh, A., Upadhyay, V., Upadhyay, A.K., Singh, S.M., and Panda, A.K., Microb. Cell Fact., 2015, vol. 14, no. 1, pp. 1–10.CrossRefGoogle Scholar
  4. 4.
    Peternel, S. and Liovi, M., Recent Patents Biomed. Engin., 2010, vol. 3, pp. 153–161.CrossRefGoogle Scholar
  5. 5.
    Diez-Gil, C., Krabbenborg, S., Garcia-Fruitos, E., Vazquez, E., Rodriguez-Carmona, E., Ratera, I., Ventosa, N., Seras-Franzoso, J., Cano-Garrido, O., Ferrer-Miralles, N., Villaverde, A., and Veciana, J., Biomaterials, 2010, vol. 31, pp. 5805–5812.CrossRefPubMedGoogle Scholar
  6. 6.
    Royt, P., WO Patent no. WO2007008769, 2007.Google Scholar
  7. 7.
    Vazquez, E., Corchero, J.L., Burgueno, J.F., Seras-Franzoso, J., Kosoy, A., Bosser, R., Mendoza, R., Martínez-Láinez, J.M., Rinas, U., Fernández, E., Ruiz-Avila, L., Gárcia-Fruitós, E., and Villaverde, A., Adv. Mater., 2012, vol. 24, pp. 1742–1747.CrossRefPubMedGoogle Scholar
  8. 8.
    Istivan, T.S. and Coloe, P.J., Microbiology, 2006, vol. 152, pp. 1263–1274.CrossRefPubMedGoogle Scholar
  9. 9.
    Ramrakhiani, L. and Chand, S., Appl. Biochem. Biotechnol., 2011, vol. 164, pp. 991–1022.CrossRefPubMedGoogle Scholar
  10. 10.
    Dekker, N., Merck, K., Tommassen, J., and Verheij, H.M., Eur. J. Biochem., 1995, vol. 232, pp. 214–219.CrossRefPubMedGoogle Scholar
  11. 11.
    Frankel, S., Sohn, R., and Leinwand, L., Proc. Natl. Acad. Sci. U. S. A., 1991, vol. 88, pp. 1192–1196.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gupta, A., Lyer, B.R., Chaturvedi, D., Maurya, S.R., and Mahalakshmi, R., RSC Adv., 2015, vol. 5, pp. 1227–1234.CrossRefGoogle Scholar
  13. 13.
    Upadhyay, A.K., Murmu, F., Singh, A., and Panda, A.K., PLoS One, 2012, vol. 7, p. e33951.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Singh, A., Upadhyay, V., Upadhyay, A.K., Singh, S.M., and Panda, A.K., Microb. Cell Fact., 2015, vol. 14, no. 1, pp. 1–10.CrossRefGoogle Scholar
  15. 15.
    Klunk, W.E., Pettegrew, J.W., and Abraham, D.J., J. Histochem. Cytochem., 1989, vol. 37, pp. 1273–1281.CrossRefPubMedGoogle Scholar
  16. 16.
    Miles, A.J. and Wallace, B.A., Chem. Soc. Rev., 2016, vol. 45, pp. 4859–4872.CrossRefPubMedGoogle Scholar
  17. 17.
    Sreerama, N., Venyaminov, S.Y., and Woody, R.W., Protein Sci., 1999, vol. 8, pp. 370–380.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Montserret, R., McLeish, M.J., Böckmann, A., Geourjon, C., and Penin, F., Biochemistry, 2000, vol. 39, no. 29, pp. 8362–8373.CrossRefPubMedGoogle Scholar
  19. 19.
    Bakholdina, S.I., Tischenko, N.M., Sidorin, E.V., Isaeva, M.P., Likhatskaya, G.N., Dmitrenok, P.S., Kim, N.Yu., Chernikov, O.V., and Solov’eva, T.F., Biochemistry (Moscow), 2016, vol. 81, no. 1, pp. 47–57.CrossRefGoogle Scholar
  20. 20.
    Pedretti, A., Villa, L., and Vistoli, G., J. Mol. Grap., 2002, vol. 21, pp. 47–49.CrossRefGoogle Scholar
  21. 21.
    Frishman, D. and Argos, P., Proteins, 1995, vol. 23, pp. 566–579.CrossRefPubMedGoogle Scholar
  22. 22.
    Provencher, C.W. and Glöcker, J., Biochemistry, 1981, vol. 20, no. 1, pp. 34–37.CrossRefGoogle Scholar
  23. 23.
    Laemmli, U.K., Nature, 1970, vol. 227, pp. 680–685.CrossRefPubMedGoogle Scholar
  24. 24.
    Tsai, C.M. and Frasch, C.E., Anal. Biochem., 1982, vol. 119, pp. 115–119.CrossRefPubMedGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2018

Authors and Affiliations

  • S. I. Bakholdina
    • 1
  • E. V. Sidorin
    • 1
  • V. A. Khomenko
    • 1
  • M. P. Isaeva
    • 1
  • N. Yu. Kim
    • 1
  • E. P. Bystritskaya
    • 1
  • E. A. Pimenova
    • 1
    • 2
  • T. F. Solov’eva
    • 1
  1. 1.Yelyakov Pacific Institute of Bioorganic Chemistry, Far East BranchRussian Academy of SciencesVladivostokRussia
  2. 2.National Scientific Center of Marine Biology, Far East BranchRussian Academy of SciencesVladivostokRussia

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