Advertisement

Applied Biochemistry and Biotechnology

, Volume 163, Issue 1, pp 80–89 | Cite as

Expression and Secretion of a CB4-1 scFv–GFP Fusion Protein by Fission Yeast

  • Julia Maria Naumann
  • Gabriele Küttner
  • Matthias BureikEmail author
Article

Abstract

There is a rapidly growing demand for fluorescent single-chain Fv (scFv) antibody fragments for many applications. Yeasts have developed into attractive hosts for recombinant production of these functionalized proteins because they provide several advantages over prokaryotes and higher eukaryotes as expression systems, e.g., being capable of high-level secretion of heterologous proteins. In this study, we report Schizosaccharomyces pombe as a new host organism for secretory production of scFv-green fluorescent protein (GFP) fusions and compare it with previously described yeast expression systems. We cloned a plasmid for the expression and secretion of the anti-p24 (human immunodeficiency virus 1) CB4-1 scFv fused to GFP. After expression of the scFv–GFP fused to an N-terminal Cpy1 secretion signal sequence, fluorescence microscopy of living yeast cells indicated that the heterologous protein entered the secretory pathway. Western blot analysis of cell-free culture supernatants confirmed that the scFv–GFP was efficiently secreted with yields up to 5 mg/L. In addition, fluorescence measurements of culture supernatants demonstrated that the GFP moiety of the scFv–GFP protein is fully functional after secretion. Our data suggest that S. pombe has the potential for being used as alternative expression host in recombinant antibody fragment production by ensuring efficient protein processing and secretion.

Keywords

Fission yeast (Schizosaccharomyces pombeHeterologous protein production Secretion Cpy1 Recombinant antibodies Single-chain Fv (scFv) fragments CB4-1 Green fluorescent protein (GFP) Functionalized scFv 

References

  1. 1.
    Alting-Mees, M. A., Risseeuw, E. P., Liu, E., Desautels, M., Crosby, W. A., & Hemmingsen, S. M. (2006). Methods in Molecular Biology, 313, 97–105.Google Scholar
  2. 2.
    Cao, M., Cao, P., Yan, H., Ren, F., Lu, W., Hu, Y., et al. (2008). Applied Microbiology and Biotechnology, 79, 423–431.CrossRefGoogle Scholar
  3. 3.
    Casey, J. L., Coley, A. M., Tilley, L. M., & Foley, M. (2000). Protein Engineering, 13, 445–452.CrossRefGoogle Scholar
  4. 4.
    Didier, P., Weiss, E., Sibler, A. P., Philibert, P., Martineau, P., Bigot, J. Y., et al. (2008). Biochemical and Biophysical Research Communications, 366, 878–884.CrossRefGoogle Scholar
  5. 5.
    Hink, M. A., Griep, R. A., Borst, J. W., van Hoek, A., Eppink, M. H., Schots, A., et al. (2000). The Journal of Biological Chemistry, 275, 17556–17560.CrossRefGoogle Scholar
  6. 6.
    Huang, D., & Shusta, E. V. (2006). Applied and Environmental Microbiology, 72, 7748–7759.CrossRefGoogle Scholar
  7. 7.
    Ishikiriyama, M., Nishina, T., Kato, T., Ueda, H., & Park, E. Y. (2009). Journal of Bioscience and Bioengineering, 107, 67–72.CrossRefGoogle Scholar
  8. 8.
    Lu, M., Gong, X. G., Yu, H., & Li, J. Y. (2005). Journal of Zhejiang University. Science B, 6, 832–837.CrossRefGoogle Scholar
  9. 9.
    Morino, K., Katsumi, H., Akahori, Y., Iba, Y., Shinohara, M., Ukai, Y., et al. (2001). Journal of Immunological Methods, 257, 175–184.CrossRefGoogle Scholar
  10. 10.
    Panjideh, H., Coelho, V., Dernedde, J., Fuchs, H., Keilholz, U., Thiel, E., et al. (2008). Bioprocess and Biosystems Engineering, 31, 559–568.CrossRefGoogle Scholar
  11. 11.
    Petrausch, U., Dernedde, J., Coelho, V., Panjideh, H., Frey, D., Fuchs, H., et al. (2007). Protein Engineering, Design & Selection, 20, 583–590.CrossRefGoogle Scholar
  12. 12.
    Schwalbach, G., Sibler, A. P., Choulier, L., Deryckere, F., & Weiss, E. (2000). Protein Expression and Purification, 18, 121–132.CrossRefGoogle Scholar
  13. 13.
    Peipp, M., Saul, D., Barbin, K., Bruenke, J., Zunino, S. J., Niederweis, M., et al. (2004). Journal of Immunological Methods, 285, 265–280.CrossRefGoogle Scholar
  14. 14.
    Worn, A., & Pluckthun, A. (2001). Journal of Molecular Biology, 305, 989–1010.CrossRefGoogle Scholar
  15. 15.
    Arbabi-Ghahroudi, M., Tanha, J., & MacKenzie, R. (2005). Cancer and Metastasis Reviews, 24, 501–519.CrossRefGoogle Scholar
  16. 16.
    Brys, R., Nelles, L., van der Schueren, E., Silvestre, N., Huylebroeck, D., & Remacle, J. E. (1998). DNA and Cell Biology, 17, 349–358.CrossRefGoogle Scholar
  17. 17.
    Jones, R. H., Moreno, S., Nurse, P., & Jones, N. C. (1988). Cell, 53, 659–667.CrossRefGoogle Scholar
  18. 18.
    Kaufer, N. F., Simanis, V., & Nurse, P. (1985). Nature, 318, 78–80.CrossRefGoogle Scholar
  19. 19.
    Remacle, J. E., Albrecht, G., Brys, R., Braus, G. H., & Huylebroeck, D. (1997). The EMBO Journal, 16, 5722–5729.CrossRefGoogle Scholar
  20. 20.
    Russell, P., & Nurse, P. (1986). Cell, 45, 781–782.CrossRefGoogle Scholar
  21. 21.
    Chappell, T. G., & Warren, G. (1989). The Journal of Cell Biology, 109, 2693–2702.CrossRefGoogle Scholar
  22. 22.
    Giga-Hama, Y., Tohda, H., Okada, H., Owada, M. K., Okayama, H., & Kumagai, H. (1994). Biotechnology (New York), 12, 400–404.CrossRefGoogle Scholar
  23. 23.
    Moreno, S., Sanchez, Y., & Rodriguez, L. (1990). The Biochemical Journal, 267, 697–702.Google Scholar
  24. 24.
    Parodi, A. J. (1999). Biochimica et Biophysica Acta, 1426, 287–295.Google Scholar
  25. 25.
    Takegawa, K., Tohda, H., Sasaki, M., Idiris, A., Ohashi, T., Mukaiyama, H., et al. (2009). Biotechnology and Applied Biochemistry, 53, 227–235.CrossRefGoogle Scholar
  26. 26.
    Kjærulff, S., & Jensen, M. R. (2005). Biochemical and Biophysical Research Communications, 336, 974–982.CrossRefGoogle Scholar
  27. 27.
    Winkler, K., Kramer, A., Kuttner, G., Seifert, M., Scholz, C., Wessner, H., et al. (2000). Journal of Immunology, 165, 4505–4514.Google Scholar
  28. 28.
    Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: A laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Press.Google Scholar
  29. 29.
    Craven, R. A., Griffiths, D. J., Sheldrick, K. S., Randall, R. E., Hagan, I. M., & Carr, A. M. (1998). Gene, 221, 59–68.CrossRefGoogle Scholar
  30. 30.
    Basi, G., Schmid, E., & Maundrell, K. (1993). Gene, 123, 131–136.CrossRefGoogle Scholar
  31. 31.
    Schmidt, T. G., Koepke, J., Frank, R., & Skerra, A. (1996). Journal of Molecular Biology, 255, 753–766.CrossRefGoogle Scholar
  32. 32.
    Dragan, C. A., Zearo, S., Hannemann, F., Bernhardt, R., & Bureik, M. (2005). FEMS Yeast Research, 5, 621–625.CrossRefGoogle Scholar
  33. 33.
    Forsburg, S. L., & Rhind, N. (2006). Yeast, 23, 173–183.CrossRefGoogle Scholar
  34. 34.
    Losson, R., & Lacroute, F. (1983). Cell, 32, 371–377.CrossRefGoogle Scholar
  35. 35.
    Suga, M., & Hatakeyama, T. (2005). Yeast, 22, 799–804.CrossRefGoogle Scholar
  36. 36.
    Kortmann, H., Kurth, F., Blank, L. M., Dittrich, P. S., & Schmid, A. (2009). Lab on a Chip, 9, 3047–3049.CrossRefGoogle Scholar
  37. 37.
    Braspenning, J., Meschede, W., Marchini, A., Muller, M., Gissmann, L., & Tommasino, M. (1998). Biochemical and Biophysical Research Communications, 245, 166–171.CrossRefGoogle Scholar
  38. 38.
    Eiden-Plach, A., Zagorc, T., Heintel, T., Carius, Y., Breinig, F., & Schmitt, M. J. (2004). Applied and Environmental Microbiology, 70, 961–966.CrossRefGoogle Scholar
  39. 39.
    Giga-Hama, Y. (1997). Fission yeast Schizosaccharomyces pombe: An attractive host for heterologous protein production. In Y. Giga-Hama & H. Kumagai (Eds.), Foreign gene expression in fission yeast Schizosaccharomyces pombe (pp. 3–28). Berlin: Springer.Google Scholar
  40. 40.
    Heintel, T., Zagorc, T., & Schmitt, M. J. (2001). Applied Microbiology and Biotechnology, 56, 165–172.CrossRefGoogle Scholar
  41. 41.
    Ciofalo, V., Barton, N., Kretz, K., Baird, J., Cook, M., & Shanahan, D. (2003). Regulatory Toxicology and Pharmacology, 37, 286–292.CrossRefGoogle Scholar
  42. 42.
    Okada, H., Sekiya, T., Yokoyama, K., Tohda, H., Kumagai, H., & Morikawa, Y. (1998). Applied Microbiology and Biotechnology, 49, 301–308.CrossRefGoogle Scholar
  43. 43.
    Okada, H., Tada, K., Sekiya, T., Yokoyama, K., Takahashi, A., Tohda, H., et al. (1998). Applied and Environmental Microbiology, 64, 555–563.Google Scholar
  44. 44.
    Kjærulff, S., Muller, S., & Jensen, M. R. (2005). Biochemical and Biophysical Research Communications, 338, 1853–1859.CrossRefGoogle Scholar
  45. 45.
    Wentz, A. E., & Shusta, E. V. (2008). Biotechnology Progress, 24, 748–756.CrossRefGoogle Scholar
  46. 46.
    Idiris, A., Bi, K., Tohda, H., Kumagai, H., & Giga-Hama, Y. (2006). Yeast, 23, 83–99.CrossRefGoogle Scholar
  47. 47.
    Idiris, A., Tohda, H., Bi, K. W., Isoai, A., Kumagai, H., & Giga-Hama, Y. (2006). Applied Microbiology and Biotechnology, 73, 404–420.CrossRefGoogle Scholar
  48. 48.
    Idiris, A., Tohda, H., Sasaki, M., Okada, K., Kumagai, H., Giga-Hama, Y., et al. (2010). Applied Microbiology and Biotechnology, 85, 667–677.CrossRefGoogle Scholar
  49. 49.
    Mukaiyama, H., Tohda, H., & Takegawa, K. (2009). Applied Microbiology and Biotechnology, 86, 1135–1143.CrossRefGoogle Scholar
  50. 50.
    Mukaiyama, H., Giga-Hama, Y., Tohda, H., & Takegawa, K. (2009). Applied Microbiology and Biotechnology, 85, 155–164.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Julia Maria Naumann
    • 1
  • Gabriele Küttner
    • 2
  • Matthias Bureik
    • 1
    Email author
  1. 1.PomBioTech GmbHSaarbrückenGermany
  2. 2.Institute of Biochemistry, Medical DepartmentHumboldt UniversityBerlinGermany

Personalised recommendations