Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1997 Feb;63(2):615–620. doi: 10.1128/aem.63.2.615-620.1997

Comparison of cell wall proteins of Saccharomyces cerevisiae as anchors for cell surface expression of heterologous proteins.

J M Van der Vaart 1, R te Biesebeke 1, J W Chapman 1, H Y Toschka 1, F M Klis 1, C T Verrips 1
PMCID: PMC168351  PMID: 9023939

Abstract

The carboxyl-terminal regions of five cell wall proteins (Cwp1p, Cwp2p, Ag alpha 1p, Tip1p, and Flo1p) and three potential cell wall proteins (Sed1p, YCR89w, and Tir1p) all proved capable of immobilizing alpha-galactosidase in the cell wall of Saccharomyces cerevisiae. The fraction of the total amount of fusion protein that was localized to the cell wall varied depending on the anchor domain used. The highest proportion of cell wall incorporation was achieved with Cwp2p, Ag alpha 1p, or Sed1p as an anchor. Although 80% of these fusion proteins were incorporated in the cell wall, the total production of alpha-galactosidase-Ag alpha 1p was sixfold lower than that of alpha-galactosidase-Cwp2p and eightfold lower than that of alpha-galactosidase-Sed1p. Differences in mRNA levels were not responsible for this discrepancy, nor was an intracellular accumulation of alpha-galactosidase-Ag alpha 1p detectable. A lower translation efficiency of the alpha-galactosidase-AG alpha 1 fusion construct is most likely to be responsible for the low level of protein production. alpha-Galactosidase immobilized by the carboxyl-terminal 67 amino acids of Cwp2p was most effective in the hydrolysis of the high-molecular-weight substrate guar gum from Cyamopsis tetragonoloba. This indicates that the use of a large anchoring domain does not necessarily result in a better exposure of the immobilized enzyme to the exterior of the yeast cell.

Full Text

The Full Text of this article is available as a PDF (1.3 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Agterberg M., Adriaanse H., Lankhof H., Meloen R., Tommassen J. Outer membrane PhoE protein of Escherichia coli as a carrier for foreign antigenic determinants: immunogenicity of epitopes of foot-and-mouth disease virus. Vaccine. 1990 Feb;8(1):85–91. doi: 10.1016/0264-410x(90)90184-n. [DOI] [PubMed] [Google Scholar]
  2. Cid V. J., Durán A., del Rey F., Snyder M. P., Nombela C., Sánchez M. Molecular basis of cell integrity and morphogenesis in Saccharomyces cerevisiae. Microbiol Rev. 1995 Sep;59(3):345–386. doi: 10.1128/mr.59.3.345-386.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Erhart E., Hollenberg C. P. The presence of a defective LEU2 gene on 2 mu DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol. 1983 Nov;156(2):625–635. doi: 10.1128/jb.156.2.625-635.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Francisco J. A., Earhart C. F., Georgiou G. Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2713–2717. doi: 10.1073/pnas.89.7.2713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Georgiou G., Poetschke H. L., Stathopoulos C., Francisco J. A. Practical applications of engineering gram-negative bacterial cell surfaces. Trends Biotechnol. 1993 Jan;11(1):6–10. doi: 10.1016/0167-7799(93)90068-K. [DOI] [PubMed] [Google Scholar]
  6. Gunneriusson E., Samuelson P., Uhlen M., Nygren P. A., Stähl S. Surface display of a functional single-chain Fv antibody on staphylococci. J Bacteriol. 1996 Mar;178(5):1341–1346. doi: 10.1128/jb.178.5.1341-1346.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hanski E., Horwitz P. A., Caparon M. G. Expression of protein F, the fibronectin-binding protein of Streptococcus pyogenes JRS4, in heterologous streptococcal and enterococcal strains promotes their adherence to respiratory epithelial cells. Infect Immun. 1992 Dec;60(12):5119–5125. doi: 10.1128/iai.60.12.5119-5125.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hardwick K. G., Boothroyd J. C., Rudner A. D., Pelham H. R. Genes that allow yeast cells to grow in the absence of the HDEL receptor. EMBO J. 1992 Nov;11(11):4187–4195. doi: 10.1002/j.1460-2075.1992.tb05512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harmsen M. M., Langedijk A. C., van Tuinen E., Geerse R. H., Raué H. A., Maat J. Effect of a pmr 1 disruption and different signal sequences on the intracellular processing and secretion of Cyamopsis tetragonoloba alpha-galactosidase by Saccharomyces cerevisiae. Gene. 1993 Mar 30;125(2):115–123. doi: 10.1016/0378-1119(93)90318-w. [DOI] [PubMed] [Google Scholar]
  10. Harrison J. L., Taylor I. M., O'Connor C. D. Presentation of foreign antigenic determinants at the bacterial cell surface using the TraT lipoprotein. Res Microbiol. 1990 Sep-Oct;141(7-8):1009–1012. doi: 10.1016/0923-2508(90)90142-d. [DOI] [PubMed] [Google Scholar]
  11. Hedegaard L., Klemm P. Type 1 fimbriae of Escherichia coli as carriers of heterologous antigenic sequences. Gene. 1989 Dec 21;85(1):115–124. doi: 10.1016/0378-1119(89)90471-x. [DOI] [PubMed] [Google Scholar]
  12. Kapteyn J. C., Montijn R. C., Vink E., de la Cruz J., Llobell A., Douwes J. E., Shimoi H., Lipke P. N., Klis F. M. Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked beta-1,3-/beta-1,6-glucan heteropolymer. Glycobiology. 1996 Apr;6(3):337–345. doi: 10.1093/glycob/6.3.337. [DOI] [PubMed] [Google Scholar]
  13. Klis F. M. Review: cell wall assembly in yeast. Yeast. 1994 Jul;10(7):851–869. doi: 10.1002/yea.320100702. [DOI] [PubMed] [Google Scholar]
  14. Little M., Fuchs P., Breitling F., Dübel S. Bacterial surface presentation of proteins and peptides: an alternative to phage technology? Trends Biotechnol. 1993 Jan;11(1):3–5. doi: 10.1016/0167-7799(93)90067-J. [DOI] [PubMed] [Google Scholar]
  15. Lopes T. S., Klootwijk J., Veenstra A. E., van der Aar P. C., van Heerikhuizen H., Raúe H. A., Planta R. J. High-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae: a new vector for high-level expression. Gene. 1989 Jul 15;79(2):199–206. doi: 10.1016/0378-1119(89)90202-3. [DOI] [PubMed] [Google Scholar]
  16. Lu C. F., Montijn R. C., Brown J. L., Klis F., Kurjan J., Bussey H., Lipke P. N. Glycosyl phosphatidylinositol-dependent cross-linking of alpha-agglutinin and beta 1,6-glucan in the Saccharomyces cerevisiae cell wall. J Cell Biol. 1995 Feb;128(3):333–340. doi: 10.1083/jcb.128.3.333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marguet D., Guo X. J., Lauquin G. J. Yeast gene SRP1 (serine-rich protein). Intragenic repeat structure and identification of a family of SRP1-related DNA sequences. J Mol Biol. 1988 Aug 5;202(3):455–470. doi: 10.1016/0022-2836(88)90278-1. [DOI] [PubMed] [Google Scholar]
  18. Montijn R. C., van Rinsum J., van Schagen F. A., Klis F. M. Glucomannoproteins in the cell wall of Saccharomyces cerevisiae contain a novel type of carbohydrate side chain. J Biol Chem. 1994 Jul 29;269(30):19338–19342. [PubMed] [Google Scholar]
  19. Newton S. M., Jacob C. O., Stocker B. A. Immune response to cholera toxin epitope inserted in Salmonella flagellin. Science. 1989 Apr 7;244(4900):70–72. doi: 10.1126/science.2468182. [DOI] [PubMed] [Google Scholar]
  20. Oliver S. G., van der Aart Q. J., Agostoni-Carbone M. L., Aigle M., Alberghina L., Alexandraki D., Antoine G., Anwar R., Ballesta J. P., Benit P. The complete DNA sequence of yeast chromosome III. Nature. 1992 May 7;357(6373):38–46. doi: 10.1038/357038a0. [DOI] [PubMed] [Google Scholar]
  21. Pozzi G., Oggioni M. R., Manganelli R., Medaglini D., Fischetti V. A., Fenoglio D., Valle M. T., Kunkl A., Manca F. Human T-helper cell recognition of an immunodominant epitope of HIV-1 gp120 expressed on the surface of Streptococcus gordonii. Vaccine. 1994 Sep;12(12):1071–1077. doi: 10.1016/0264-410x(94)90175-9. [DOI] [PubMed] [Google Scholar]
  22. Schneewind O., Fowler A., Faull K. F. Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science. 1995 Apr 7;268(5207):103–106. doi: 10.1126/science.7701329. [DOI] [PubMed] [Google Scholar]
  23. Schreuder M. P., Brekelmans S., van den Ende H., Klis F. M. Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast. 1993 Apr;9(4):399–409. doi: 10.1002/yea.320090410. [DOI] [PubMed] [Google Scholar]
  24. Schreuder M. P., Mooren A. T., Toschka H. Y., Verrips C. T., Klis F. M. Immobilizing proteins on the surface of yeast cells. Trends Biotechnol. 1996 Apr;14(4):115–120. doi: 10.1016/0167-7799(96)10017-2. [DOI] [PubMed] [Google Scholar]
  25. Sierkstra L. N., Verbakel J. M., Verrips C. T. Analysis of transcription and translation of glycolytic enzymes in glucose-limited continuous cultures of Saccharomyces cerevisiae. J Gen Microbiol. 1992 Dec;138(12):2559–2566. doi: 10.1099/00221287-138-12-2559. [DOI] [PubMed] [Google Scholar]
  26. Teunissen A. W., Holub E., van der Hucht J., van den Berg J. A., Steensma H. Y. Sequence of the open reading frame of the FLO1 gene from Saccharomyces cerevisiae. Yeast. 1993 Apr;9(4):423–427. doi: 10.1002/yea.320090413. [DOI] [PubMed] [Google Scholar]
  27. Van Rinsum J., Klis F. M., van den Ende H. Cell wall glucomannoproteins of Saccharomyces cerevisiae mnn9. Yeast. 1991 Oct;7(7):717–726. doi: 10.1002/yea.320070707. [DOI] [PubMed] [Google Scholar]
  28. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  29. van der Vaart J. M., Caro L. H., Chapman J. W., Klis F. M., Verrips C. T. Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol. 1995 Jun;177(11):3104–3110. doi: 10.1128/jb.177.11.3104-3110.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. van der Vaart J. M., van Schagen F. S., Mooren A. T., Chapman J. W., Klis F. M., Verrips C. T. The retention mechanism of cell wall proteins in Saccharomyces cerevisiae. Wall-bound Cwp2p is beta-1,6-glucosylated. Biochim Biophys Acta. 1996 Dec 6;1291(3):206–214. doi: 10.1016/s0304-4165(96)00067-0. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES