Skip to main content
SpringerLink
Log in

Construction of a Novel Pichia pastoris Cell-Surface Display System Based on the Cell Wall Protein Pir1

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

A novel system based on Pir1 from Saccharomyces cerevisiae was developed for cell-surface display of heterologous proteins in Pichia pastoris with the alpha-factor secretion signal sequence. As a model protein, enhanced green fluorescence protein (EGFP) was fused to the N-terminal of the mature peptide of Pir1 (Pir1-a). The expression of fusion protein EGFP-Pir1-a was irregular throughout the P. pastoris cell surface per detection by confocal laser scanning microscopy. A truncated sequence containing only the internal repetitive sequences of Pir1-a (Pir1-b) was used as a new anchor protein in further study. The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface. The fluorescence intensity of the whole yeast was measured by spectrofluorometer. Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment. The results indicate that a Pir1-based system can express proteins on the surface of P. pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall. The repetitive sequences of Pir1 are required for cell wall retention, and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P. pastoris.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Abe H, Shimma Y, Jigami Y (2003) In vitro oligosaccharide synthesis using intact yeast cells that display glycosyltransferases at the cell surface through cell wall-anchored protein Pir. Glycobiology 13:87–95

    Article  PubMed  CAS  Google Scholar 

  2. Andres I, Gallardo O, Parascandola P, et al. (2005) Use of the cell wall protein Pir4 as a fusion partner for the expression of Bacillus sp. BP-7 xylanase A in Saccharomyces cerevisiae. Biotechnol Bioeng 89:690–697

    Article  PubMed  CAS  Google Scholar 

  3. Bony M, Thines-Sempoux D, Barre P, et al. (1997) Localization and cell surface anchoring of the Saccharomyces cerevisiae flocculation protein Flo1p. J Bacteriol 179:4929–4936

    PubMed  CAS  Google Scholar 

  4. Castillo L, Martinez AI, Garcera A, et al. (2003) Functional analysis of the cysteine residues and the repetitive sequence of Saccharomyces cerevisiae Pir4/Cis3: The repetitive sequence is needed for binding to the cell wall beta-1,3-glucan. Yeast 20:973–983

    Article  PubMed  CAS  Google Scholar 

  5. Choi BK, Bobrowicz P, Davidson RC, et al. (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci U S A 100:5022–5027

    Article  PubMed  CAS  Google Scholar 

  6. Du C, Chan WC, McKeithan TW, et al. (2005) Surface display of recombinant proteins on Bacillus thuringiensis spores. Appl Environ Microbiol 71:3337–3341

    Article  PubMed  CAS  Google Scholar 

  7. Ecker M, Deutzmann R, Lehle L, et al. (2006) Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage. J Biol Chem 281:11523–11529

    Article  PubMed  CAS  Google Scholar 

  8. Gemmill TR, Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426:227–237

    PubMed  CAS  Google Scholar 

  9. Gunneriusson E, Samuelson P, Uhlen M, et al. (1996) Surface display of a functional single-chain Fv antibody on staphylococci. J Bacteriol 178:1341–1346

    PubMed  CAS  Google Scholar 

  10. Kapteyn JC, Montijn RC, Vink E, et al. (1996) Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology 6:337–345

    Article  PubMed  CAS  Google Scholar 

  11. Kapteyn JC, Van Den Ende H, Klis FM (1999) The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1426:373–383

    PubMed  CAS  Google Scholar 

  12. Kondo A, Ueda M (2004) Yeast cell-surface display―Applications of molecular display. Appl Microbiol Biotechnol 64:28–40

    Article  PubMed  CAS  Google Scholar 

  13. Li Z, Xiong F, Lin Q, et al. (2001) Low-temperature increases the yield of biologically active herring antifreeze protein in Pichia pastoris. Protein Expr Purif 21:438–445

    Article  PubMed  Google Scholar 

  14. Matsumoto T, Fukuda H, Ueda M, et al. (2002) Construction of yeast strains with high cell surface lipase activity by using novel display systems based on the Flo1p flocculation functional domain. Appl Environ Microbiol 68:4517–4522

    Article  PubMed  CAS  Google Scholar 

  15. Mergler M, Wolf K, Zimmermann M (2004) Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris. Appl Microbiol Biotechnol 63:418–421

    Article  PubMed  CAS  Google Scholar 

  16. Mrsa V, Seidl T, Gentzsch M, et al. (1997) Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae. Yeast 13:1145–1154

    Article  PubMed  CAS  Google Scholar 

  17. Mrsa V, Tanner W (1999) Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall. Yeast 15:813–820

    Article  PubMed  CAS  Google Scholar 

  18. Shi H, Wen Su W (2001) Display of green fluorescent protein on Escherichia coli cell surface. Enzyme Microb Technol 28:25–34

    Article  PubMed  CAS  Google Scholar 

  19. Shimma Y, Saito F, Oosawa F, et al. (2006) Construction of a library of human glycosyltransferases immobilized in the cell wall of Saccharomyces cerevisiae. Appl Environ Microbiol 72:7003–7012

    Article  PubMed  CAS  Google Scholar 

  20. Stahl S, Uhlen M (1997) Bacterial surface display: Trends and progress. Trends Biotechnol 15:185–192

    Article  PubMed  CAS  Google Scholar 

  21. Steidler L, Viaene J, Fiers W, et al. (1998) Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Appl Environ Microbiol 64:342–345

    PubMed  CAS  Google Scholar 

  22. Sumita T, Yoko-o T, Shimma Y, et al. (2005) Comparison of cell wall localization among Pir family proteins and functional dissection of the region required for cell wall binding and bud scar recruitment of Pir1p. Eukaryot Cell 4:1872–1881

    Article  PubMed  CAS  Google Scholar 

  23. Takayama K, Suye S, Kuroda K, et al. (2006) Surface display of organophosphorus hydrolase on Saccharomyces cerevisiae. Biotechnol Prog 22:939–943

    Article  PubMed  CAS  Google Scholar 

  24. Tanino T, Fukuda H, Kondo A (2006) Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol Prog 22:989–993

    Article  PubMed  CAS  Google Scholar 

  25. Teparic R, Stuparevic I, Mrsa V (2007) Binding assay for incorporation of alkali-extractable proteins in the Saccharomyces cerevisiae cell wall. Yeast 24:259–266

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This project was supported by a key fund from the Chinese Education Ministry (2005) and a grant from the National Natural Science Foundation of China (Grant No. 30470399).

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Technology, Life Science School, Shandong University, Jinan, 250100, PRC

    Qingjie Wang, Lei Li, Min Chen, Qingsheng Qi & Peng George Wang

Authors
  1. Qingjie Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lei Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Min Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qingsheng Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peng George Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peng George Wang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wang, Q., Li, L., Chen, M. et al. Construction of a Novel Pichia pastoris Cell-Surface Display System Based on the Cell Wall Protein Pir1. Curr Microbiol 56, 352–357 (2008). https://doi.org/10.1007/s00284-007-9089-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00284-007-9089-1

Keywords

Search

Navigation