Applied Microbiology and Biotechnology

, Volume 67, Issue 3, pp 364–369 | Cite as

Secretory expression of heterologous protein in Kluyveromyces cicerisporus

  • X. P. Cai
  • J. Zhang
  • H.-Y. Yuan
  • Z.-A. Fang
  • Y.-Y. Li
Applied Genetics and Molecular Biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

To explore the potential of heterologous protein expression in Kluyveromyces cicerisporus, three expression plasmids, pUK1-PIT, pUKD-PIT and pUKD-S-PIT, based on the vector pUK1 or pUKD were constructed and transformed, respectively, into yeast strain K. cicerisporus Y179U. Human interferon α-2a, used as an example protein, was successfully expressed and secreted by transformant Y179U/pUKD-PIT and Y179U/pUKD-S-PIT. In the flask culture, strain Y179U/pUKD-S-PIT could express interferon at 60 mg/l. The stability of plasmid pUKD-S-PIT in the host was higher than that of pUKD-PIT. This was consistent with their expression levels of interferon. There were two interferon-related bands found by Western blotting analysis. The possible reason for this is discussed.

Keywords

Interferon Expression Plasmid Heterologous Protein Kluyveromyces Plasmid Stability 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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Notes

Acknowledgement

This project was granted financial support from the China Postdoctoral Science Foundation.

References

  1. Bianchi MM, Santarelli R, Frontali L (1991) Plasmid functions involved in the stable propagation of the pKD1 circular plasmid in Kluyveromyces lactis. Curr Genet 19:155–161CrossRefPubMedGoogle Scholar
  2. Fleer R, Chen XJ, Amellal N, Yeh P, Fournier A, Guinet F, Gault N, Faucher D, Folliard F, Fukuhara H (1991) High-level secretion of correctly processed recombinant human interleukin-1 beta in Kluyveromyces lactis. Gene 107:285–295CrossRefPubMedGoogle Scholar
  3. Hashimoto Y, Koyabu N, Imoto T (1998) Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors. Protein Eng 11:75–77CrossRefPubMedGoogle Scholar
  4. Hitzeman RA, Leung DW, Perry LJ, Kohr WJ, Levine HL, Goeddel DV (1983) Secretion of human interferons by yeast. Science 219:620–625PubMedGoogle Scholar
  5. Huo KK, Yu LL, Chen XJ, Li YY (1993) A stable vector for high-level expression and secretion of human interferon alpha A in yeast. Sci China B 36:557–567PubMedGoogle Scholar
  6. Jahic M, Rotticci-Mulder JC, Martinelle M, Hult K, Enfors SO (2002) Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein. Bioprocess Biosystem Eng 24:385–393CrossRefGoogle Scholar
  7. Jayaram M, Li YY, Broach JR (1983) The yeast plasmid 2mu circle encodes components required for its high copy propagation. Cell 34:95–104CrossRefPubMedGoogle Scholar
  8. Platis D, Foster GR (2003) High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli. Protein Expr Purif 31:222–230CrossRefPubMedGoogle Scholar
  9. Rossmann C, Sharp N, Allen G, Gewert D (1996) Expression and purification of recombinant, glycosylated human interferon alpha 2b in murine myeloma NSO cells. Protein Expr Purif 7:335–342CrossRefPubMedGoogle Scholar
  10. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  11. Swinkels BW, van Ooyen AJ, Bonekamp FJ (1993) The yeast Kluyveromyces lactis as an efficient host for heterologous gene expression. Antonie Van Leeuwenhoek J Serol Microbiol 64:187–201Google Scholar
  12. Wang H, Gao B, Chen P, Dong L, Li Y (1995) Analysis of heterogeneity of gene products (interferon) expressed in yeast. Sci China B 38:954–962PubMedGoogle Scholar
  13. Wen TQ, Liu F, Huo KK, Li YY (2003) Cloning and analysis of the inulinase gene from Kluyeromyces cicerisporus CBS4857. World J Microbiol Biotechnol 19:426CrossRefGoogle Scholar
  14. Xu GZ, Pan F, Huo KK, Li YY (1998) Cloning of DNA Fragments Possessing Promoter Function from Kluyveromyces cicerisporus. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai) 30:35–40Google Scholar
  15. Zhang J, Yuan H, Wen T, Xu F, Di Y, Huo K, Li YY (2003) Cloning of the KcURA3 gene and development of a transformation system for Kluyveromyces cicerisporus. Appl Microbiol Biotechnol 62:387–391CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • X. P. Cai
    • 1
  • J. Zhang
    • 1
  • H.-Y. Yuan
    • 1
  • Z.-A. Fang
    • 1
  • Y.-Y. Li
    • 1
  1. 1.Institute of Genetics, State Key Laboratory of Genetic Engineering, School of Life SciencesFudan UniversityShanghaiChina

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