Skip to main content
SpringerLink
Log in

Flow cytometric analysis of human lysozyme production in recombinantSaccharomyces cerevisiae

  • Published:
Biotechnology and Bioprocess Engineering Aims and scope Submit manuscript

Abstract

Flow cytometric techniques were used to investigate cell size, protein content and cell cycle behavior of recombinantSaccharomyces cerevisiae strains producing human lysozyme (HLZ). Two different signal sequences, the native yeastMFα1 signal sequence and the rat α-amylase signal sequence, were used for secretion of HLZ. The strain containing the rat α-amylase signal sequence showed a higher level of internal lysozyme and lower specific growth rates. Flow cytometric analysis of the total protein content and cell size showed the strain harboring the native yeast signal sequence had a higher total protein content than the strain containing the rat α-amylase signal sequence. Cell cycle analysis indicated that the two lysozyme producing recombinant strains had an increased number of cells in the G2+M phase of the yeast cell cycle compared with the host strain SEY2102.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Cho, K. M. and Y. J. Yoo (1999) Novel SSF proces for ethanol production from microcrystalline cellulose using the δ-integrated recombinant yeast,Saccharomyces cerevisiae L2612δGC.J. Microbiol. Biotechnol. 9: 340–345.

    CAS  Google Scholar 

  2. Chung, B. H., W. K. Kim, K. J. Rao, C. H. Kim, and S. K. Rhee (1999) Downstream processing of recombinant hirudin produced inSaccharomyces cerevisiae.J. Microbiol. Biotechnol. 9: 179–183.

    CAS  Google Scholar 

  3. Kim, M. D., Y. J. Yoo, S. K. Rhee, and J. H. Seo (2001) Enhanced transformation efficiency of an anticoagulant hirudin gene intoSaccharomyces cerevisiae by a double δ-sequence.J. Microbiol. Biotechnol. 11: 61–64.

    CAS  Google Scholar 

  4. Koo, J. H., S. Y. Kim, Y. C. Park, N. S. Han, and J. H. Seo (1998) Invertase production by fed-batch fermentations of recombinantSaccharomyces cerevisiae.J. Microbiol. Biotechnol. 8: 203–207.

    CAS  Google Scholar 

  5. Marten, M. R., N. S. Han, J. B. Park, and J. H. Seo (1999) Analysis of secretion behavior of human lysozyme from recombinantSaccharomyces cerevisiae.J. Microbiol. Biotechnol. 9: 576–681.

    CAS  Google Scholar 

  6. Park, J. N., D. J. Shin, H. O. Kim, D. H. Kim, H. B. Lee, S. B. Chun, and S. Bai (1999) Expression ofSchwanniomyces occidentalis α-amylase geneSaccharomyces cerevisiae var.diastaticus.J. Microbiol. Biotechnol. 9: 668–671.

    CAS  Google Scholar 

  7. Peterson, M. S., A. Y. Patkar, and J. H. Seo (1992) Flow cytometric analysis of total protein content and size distribution of recombinantSaccharomyces cerevisiae.Biotechnol. Tech. 6: 203–206.

    Article  CAS  Google Scholar 

  8. Romanos, M. A., C. A. Scorer, and J. J. Clare (1992) Foreign gene expression in yeast: A review.Yeast 8: 423–488.

    Article  CAS  Google Scholar 

  9. Seo, J. H. and J. E. Bailey (1985) Effects of recombinant plasmid content on growth properties and cloned gene product formation inE. coli.Biotechnol. Bioeng. 27: 1668–1674.

    Article  CAS  Google Scholar 

  10. Shapiro, H. M. (1995)Practical Flow Cytometry. 3rd ed., p. 339. Wiley-Liss, New York, USA.

    Google Scholar 

  11. Alberghina, L. and D. Porro (1993) Quantitative flow cytometry: analysis of protein distributions in budding yeast.Yeast 9: 815–823.

    Article  CAS  Google Scholar 

  12. Compagno, C., D. Porro, S. Radice, E. Martegani, and B. M. Ranzi (1996) Selection of yeast cells with a higher plasmid copy number in aSaccharomyces cerevisiae autoselection system.Yeast 12: 199–205.

    Article  CAS  Google Scholar 

  13. Eitzman, P. D. and E. Srienc (1991) Dynamics of activation of a galactose-inducible promoter inSaccharomyces cerevisiae.J. Biotechnol. 21: 63–81.

    Article  CAS  Google Scholar 

  14. Li, F., P. L. Flanary, D. C. Altieri, and H. G. Dohlman (2000) Cell division regulation byBIR1, a member of the inhibitor of apoptosis family in yeast.J. Biol. Chem. 275: 6707–6711.

    Article  CAS  Google Scholar 

  15. Mateus, C. and S. V. Avery (2000) Destabilized green fluorescence protein for monitoring dynamic changes in yeast gene expression with flow cytometry.Yeast 16: 1313–1323.

    Article  CAS  Google Scholar 

  16. Porro, D., C. Smeraldi, E. martegani, B. M. Ranzi, and L. Alberghina (1994) Flow-cytometric determination of the respiratory activity in growingSaccharomyces cerevisiae populations.Biotechnol. Prog. 10: 193–197.

    Article  CAS  Google Scholar 

  17. Porro, D., E. Martegani, B. M. Ranzi, and L. Alberghina (1997) Identification of different daughter and parent subpopulations in an asynchronously growingSaccharomyces cerevisiae population.Res. Microbiol. 148: 205–215.

    Article  CAS  Google Scholar 

  18. Rodriguez, F., L. Popolo, M. Vai, E. Lacana, and L. Alberghina (1990) Changes in the protein synthesis pattern during a nutritional shift-down transition inSaccharomyces cerevisiae.Exp. Cell. Res. 187: 315–319.

    Article  CAS  Google Scholar 

  19. Schlesinger, M. B. and T. Formosa (2000) POB3 is required for both transcription and replication in yeastSaccharomyces cerevisiae.Genetics 155: 1593–1606.

    CAS  Google Scholar 

  20. Smeraldi, C., E. Berardi, and D. Porro (1994) Monitoring of peroxisome induction and degradation by flow cytometric analysis ofHansenula polymorpha cells growing on methanol and glucose media: cell volume, refractive index and FITC retention.Microbiology 140: 3161–3166.

    Article  CAS  Google Scholar 

  21. Srienc F. and B. S. Dien (1992) Kinetics of the cell cycle ofSaccharomyces cerevisiae.Ann. NY Acad. Sci. 665: 59–71.

    Article  CAS  Google Scholar 

  22. Vanoni, M., M. Lotti, and L. Alberghina (1989) Expression of clonedSaccharomyces diastaticus glucoamylase under natural and inducible promoters.Biochim. Biophys. Acta 1008: 168–176.

    CAS  Google Scholar 

  23. Blake, C. C. F., D. F. Koenig, G. A. Mair, A. L. T. North, D. C. Philips, and N. R. Sarma (1965) Structure of hen egg white lysozyme.Nature 206: 757–761.

    Article  CAS  Google Scholar 

  24. Castaman, M. J., W. Spevak, G. R. Adolf, E. Chlebowicz-Sledziewska, and A. Sledziewska (1988) Cloning of human lysozyme gene and expression in the yeastSaccharomyces cerevisiae.Gene 66: 223–234.

    Article  Google Scholar 

  25. Morsky, P. (1983) Turbidimetric determination of lysozyme withMicrococcus lysodeikticus cells: Reexamination of reaction conditions.Anal. Biochem. 128: 77–85.

    Article  CAS  Google Scholar 

  26. Chapman C. and W. Bartley (1968) The kinetics of enzyme changes in yeast under conditions that cause the loss of mitochondria.Biochem. J. 107: 455–459.

    CAS  Google Scholar 

  27. Polakis E. S., W. Bartley, and G. A. Meek (1964) Changes in the structure and enzyme activity ofSaccharomyces cerevisiae in response to changes in the environment.Biochem. J. 90: 369–374.

    CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Food Science and Technology and Research Center for New Bio-Materials in Agriculture, Seoul National University, 441-744, Suwon, Korea

    Myoung-Dong Kim, Ki-Cheol Han & Jin-Ho Seo

  2. Signal Pharmaceuticals Inc., 92121, San Diego, CA, USA

    Marvin S. Peterson

  3. Ministry of Commerce, Industry and Energy, 427-010, Kwacheon, Korea

    Ji-Hyun Kim

Authors
  1. Marvin S. Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Myoung-Dong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ki-Cheol Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ji-Hyun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jin-Ho Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin-Ho Seo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Peterson, M.S., Kim, MD., Han, KC. et al. Flow cytometric analysis of human lysozyme production in recombinantSaccharomyces cerevisiae . Biotechnol. Bioprocess Eng. 7, 52–55 (2002). https://doi.org/10.1007/BF02935880

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02935880

Keywords

Search

Navigation