Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Apr;175(7):2102–2106. doi: 10.1128/jb.175.7.2102-2106.1993

Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

V Mrsa 1, F Klebl 1, W Tanner 1
PMCID: PMC204315  PMID: 8458852

Abstract

One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.

Full text

PDF
2102

Images in this article

2103Image
on p.2103
2104Image
on p.2104
2105Image
on p.2105

Selected References

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

  1. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  2. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  3. Emr S. D., Schekman R., Flessel M. C., Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  5. Klebl F., Tanner W. Molecular cloning of a cell wall exo-beta-1,3-glucanase from Saccharomyces cerevisiae. J Bacteriol. 1989 Nov;171(11):6259–6264. doi: 10.1128/jb.171.11.6259-6264.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kuranda M. J., Robbins P. W. Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 May;84(9):2585–2589. doi: 10.1073/pnas.84.9.2585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Mrsa V., Ugarković T., Barbarić S. Binding of Saccharomyces cerevisiae extracellular proteins to glucane. Arch Biochem Biophys. 1992 Aug 1;296(2):569–574. doi: 10.1016/0003-9861(92)90612-z. [DOI] [PubMed] [Google Scholar]
  10. Nebreda A. R., Villa T. G., Villanueva J. R., del Rey F. Cloning of genes related to exo-beta-glucanase production in Saccharomyces cerevisiae: characterization of an exo-beta-glucanase structural gene. Gene. 1986;47(2-3):245–259. doi: 10.1016/0378-1119(86)90068-5. [DOI] [PubMed] [Google Scholar]
  11. Roitsch T., Lehle L. Post-translational translocation of polypeptides across the mammalian endoplasmic reticulum membrane is size and ribosome dependent. Eur J Biochem. 1988 Jul 1;174(4):699–705. doi: 10.1111/j.1432-1033.1988.tb14154.x. [DOI] [PubMed] [Google Scholar]
  12. Shinshi H., Wenzler H., Neuhaus J. M., Felix G., Hofsteenge J., Meins F. Evidence for N- and C-terminal processing of a plant defense-related enzyme: Primary structure of tobacco prepro-beta-1,3-glucanase. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5541–5545. doi: 10.1073/pnas.85.15.5541. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES