Advertisement

Biochemistry (Moscow)

, 74:1187 | Cite as

Production of biologically active human myelocytokines in plants

  • A. S. ZverevaEmail author
  • L. E. Petrovskaya
  • A. V. Rodina
  • O. Y. Frolova
  • P. A. Ivanov
  • L. N. Shingarova
  • T. V. Komarova
  • Y. L. Dorokhov
  • D. A. Dolgikh
  • M. P. Kirpichnikov
  • J. G. Atabekov
Accelerated Publication

Abstract

An effective system for expression of human granulocyte and granulocyte macrophage colony-stimulating factors (hG-CSF and hGM-CSF) in Nicotiana benthamiana plants was developed using viral vector based on tobacco mosaic virus infecting cruciferous plants. The genes of target proteins were cloned into the viral vector driven by actin promoter of Arabidopsis thaliana. The expression vectors were delivered into plant cells by agroinjection. Maximal synthesis rate was detected 5 days after injection and was up to 500 and 300 mg per kg of fresh leaves for hG-CSF and hGM-CSF, respectively. The yield of purified hG-CSF and hGM-CSF was 100 and 50 mg/kg of fresh leaves, respectively. Recombinant plant-made hG-CSF and hGM-CSF stimulated proliferation of murine bone marrow and human erythroleucosis TF-1 cells, respectively, at the same rate as the commercial drugs.

Key words

granulocyte colony-stimulating factor granulocyte macrophage colony-stimulating factor viral vector agroinjection 

Abbreviations

CFU-C

colony-forming units in culture

crTMV

tobacco mosaic virus infecting cruciferous plants

ED50

dose necessary for 50% stimulation of proliferation

FCS

fetal calf serum

GFP

green fluorescent protein

hG-CSF

human granulocyte colony-stimulating factor

hGM-CSF

human granulocyte macrophage colony-stimulating factor

IPTG

isopropyl-β-D-thiogalactopyranoside

phG-CSF

rhG-CSF synthesized in plants

phGM-CSF

rhGM-CSF synthesized in plants

PMSF

phenylmethylsulfonyl fluoride

rhG-CSF

recombinant hG-CSF

rhGM-CSF

recombinant hGM-CSF

References

  1. 1.
    Giddings, G., Allison, G., Brooks, D., and Carter, A. (2000) Nature Biotechnol., 18, 1151–1155.CrossRefGoogle Scholar
  2. 2.
    Giritch, A., Marillonnet, S., Engler, C., van Eldik, G., Botterman, J., Klimyuk, V., and Gleba, Y. (2006) PNAS, 103, 14645–14646.CrossRefGoogle Scholar
  3. 3.
    Dorokhov, Y., Skurat, E., Frolova, O., Gasanova, T., Smirnov, A., Zvereva, S., Ivanov, P., Ravin, N., Zamchuk, L., and Atabekov, I. (2004) Dokl. Biokhim. Biofiz., 394, 30–32.Google Scholar
  4. 4.
    Gils, M., Kandzia, R., Marillonnet, S., Klimyuk, V., and Gleba, Y. (2005) Plant Biotechnol. J., 6, 613–620.CrossRefGoogle Scholar
  5. 5.
    Mechtcheriakova, I., Eldarov, M., Nicholson, L., Shanks, M., Skryabin, K., and Lomonossoff, G. (2006) J. Virol. Meth., 131, 10–15.CrossRefGoogle Scholar
  6. 6.
    Dorokhov, Y., Sheveleva, A., Frolova, O., Komarova, T., Zvereva, A., Ivanov, P., and Atabekov, J. (2007) Tuberculosis, 87, 218–224.CrossRefPubMedGoogle Scholar
  7. 7.
    Santi, L., Giritch, A., Roy, C., Marillonnet, S., Klimyuk, V., Gleba, Y., Webb, R., Arntzen, C., and Mason, H. (2006) PNAS, 103, 861–866.CrossRefPubMedGoogle Scholar
  8. 8.
    Hiatt, A., and Pauly, M. (1989) Nature, 342, 76–78.CrossRefPubMedGoogle Scholar
  9. 9.
    Basu, S., Dunn, A., and Ward, A. (2002) Int. J. Mol. Med., 10, 3–10.PubMedGoogle Scholar
  10. 10.
    Isola, L., Scigliano, E., Skerrett, D., Shank, B., Ross, V., Najfeld, V., and Fruchtman, S. (1997) Bone Marrow Transplant., 20, 1033–1037.CrossRefPubMedGoogle Scholar
  11. 11.
    Costello, R. (1993) Acta Oncol., 32, 403–408.CrossRefPubMedGoogle Scholar
  12. 12.
    Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003) Plant J., 33, 949–956.CrossRefPubMedGoogle Scholar
  13. 13.
    Sambrook, J., and Russell, D. (2001) Molecular Cloning: A Laboratory Manual, CSHL Press.Google Scholar
  14. 14.
    Gasparian, M., Ostapchenko, V., Schulga, A., Dolgikh, D., and Kirpichnikov, M. (2003) Protein Exp. Purif., 31, 133–139.CrossRefGoogle Scholar
  15. 15.
    Shingarova, L. N., Kashyap, S. K., Petrovskaya, L. E., Petrenko, L. A., Pustoshilova, N. M., Sinichkina, S. A., and Korobko, V. G. (1998) Biotekhnologiya, 6, 24–35.Google Scholar
  16. 16.
    Petrovskaya, L. E., Kryukova, E. A., Kayushin, A. L., Rodina, A. V., Moskaleva, E. Y., and Korobko, V. G. (2002) Bioorg. Khim., 28, 440–446.Google Scholar
  17. 17.
    Laemmli, U. (1970) Nature, 227, 680–685.CrossRefPubMedGoogle Scholar
  18. 18.
    Yamamoto, A., Iwata, A., Saitoh, T., Tuchiya, K., Kanai, T., Tsujimoto, H., Hasegawa, A., Ishihama, A., and Ueda, S. (2002) Veter. Immunol. Immunopathol., 90, 169–177.CrossRefGoogle Scholar
  19. 19.
    Mayer, P. (1983) Comp. Immunol. Microbiol. Infect. Dis., 6, 171–187.CrossRefPubMedGoogle Scholar
  20. 20.
    Hilton, D., Nicola, N., Gough, N., and Metcalf, D. (1988) J. Biol. Chem., 263, 9238–9243.PubMedGoogle Scholar
  21. 21.
    Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y., Miyazono, K., Urabe, A., and Takaku, F. (1989) J. Cell Physiol., 140, 323–334.CrossRefPubMedGoogle Scholar
  22. 22.
    Sijmons, P., Dekker, B., Schrammeijer, B., Verwoerd, T., van den Elzen, P., and Hoekema, A. (1990) Nature Biotechnol., 8, 217–221.CrossRefGoogle Scholar
  23. 23.
    Edelbaum, O., Stein, D., Holland, N., Gafni, Y., Livneh, O., Novick, D., Rubinstein, M., and Sela, I. (1992) J. Interferon Res., 12, 449–453.PubMedGoogle Scholar
  24. 24.
    Kusnadi, A., Nikolov, Z., and Howard, J. (1997) Biotechnol. Bioeng., 56, 473–484.CrossRefPubMedGoogle Scholar
  25. 25.
    Stam, M., Mol, J., and Kooter, J. (1997) Annals Bot., 79, 3–12.CrossRefGoogle Scholar
  26. 26.
    Marillonnet, S., Thoeringer, C., Kandzia, R., Klimyuk, V., and Gleba, Y. (2005) Nature Biotechnol., 23, 718–723.CrossRefGoogle Scholar
  27. 27.
    Shivprasad, S., Pogue, G., Lewandowski, D., Hidalgo, J., Donson, J., Grill, L., and Dawson, W. (1999) Virology, 255, 312–323.CrossRefPubMedGoogle Scholar
  28. 28.
    Kumagai, M., Turpen, T., Weinzettl, N., Della-Cioppa, G., Turpen, A., Donson, J., Hilf, M., Grantham, G., Dawson, W., and Chow, T. (1993) PNAS, 90, 427–430.CrossRefPubMedGoogle Scholar
  29. 29.
    Marillonnet, S., Giritch, A., Gils, M., Kandzia, R., Klimyuk, V., and Gleba, Y. (2004) PNAS, 101, 6852–6857.CrossRefPubMedGoogle Scholar
  30. 30.
    Hong, S., Kwon, T., Lee, J., Jang, Y., and Yang, M. (2002) Enzyme Microb. Technol., 30, 763–767.CrossRefGoogle Scholar
  31. 31.
    Sardana, R., Alli, Z., Dudani, A., Tackaberry, E., Panahi, M., Narayanan, M., Ganz, P., and Altosaar, I. (2002) Transgen. Res., 11, 521–531.CrossRefGoogle Scholar
  32. 32.
    James, E., Wang, C., Wang, Z., Reeves, R., Shin, J., Magnuson, N., and Lee, J. (2000) Protein Exp. Purif., 19, 131–138.CrossRefGoogle Scholar
  33. 33.
    Kwon, T., Kim, Y., Lee, J., and Yang, M. (2003) Biotechnol. Lett., 25, 1571–1574.CrossRefPubMedGoogle Scholar
  34. 34.
    Wang, M., Goldstein, C., Su, W., Moore, P., and Albert, H. (2005) Transgen. Res., 14, 167–178.CrossRefGoogle Scholar
  35. 35.
    Zhou, F., Wang, M., Albert, H., Moore, P., and Zhu, Y. (2006) Appl. Microbiol. Biotechnol., 72, 756–762.CrossRefPubMedGoogle Scholar
  36. 36.
    Woestenenk, E., Hammarstrom, M., van den Berg, S., Hard, T., and Berglund, H. (2004) J. Struct. Funct. Genom., 5, 217–229.CrossRefGoogle Scholar
  37. 37.
    Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003) Plant J., 33, 949–956.CrossRefPubMedGoogle Scholar
  38. 38.
    Nicola, N. (1987) Int. J. Cell Cloning, 5, 1–15.CrossRefPubMedGoogle Scholar
  39. 39.
    Scheerlinck, J. (1999) Veter. Immunol. Immunopathol., 72, 39–44.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • A. S. Zvereva
    • 1
    Email author
  • L. E. Petrovskaya
    • 2
  • A. V. Rodina
    • 3
  • O. Y. Frolova
    • 1
  • P. A. Ivanov
    • 1
  • L. N. Shingarova
    • 2
  • T. V. Komarova
    • 4
  • Y. L. Dorokhov
    • 4
  • D. A. Dolgikh
    • 2
  • M. P. Kirpichnikov
    • 1
    • 2
  • J. G. Atabekov
    • 1
  1. 1.Faculty of BiologyLomonosov Moscow State UniversityMoscowRussia
  2. 2.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  3. 3.Moscow Institute of Medical EcologyMoscowRussia
  4. 4.Belozersky Institute of Physico-Chemical BiologyLomonosov Moscow State UniversityMoscowRussia

Personalised recommendations