Summary
Filamentous fungi have been recognized as extraordinary producers of secreted proteins and are known to produce novel proteins and enzymes through dispensable metabolic pathways. Here, methods are described for the isolation and enrichment of samples of secreted proteins from cultures of filamentous fungi for analysis by gel electrophoresis and mass spectrometry techniques. These methods can be readily applied to the study of differential protein expression and secretion and metabolic pathways in filamentous fungi by proteomic approaches.
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References
Peberdy, J. F. (1994) Protein secretion in filamentous fungi - trying to understand a highly productive black-box, Trends Biotechnol. 12, 50–7.
Conesa, A., Punt, P. J., van Luijk, N., and van den Hondel, C. A. A. J. J. (2001) The secretion pathway in filamentous fungi: A biotechnological view, Fungal. Genet. Biol. 33, 155–71.
Archer, D. B. and Peberdy, J. F. (1997) The molecular biology of secreted enzyme production by fungi, Crit. Rev. Biotechnol. 17, 273–306.
Wallis, G. L. F., Swift, R. J., Hemming, F. W., Trinci, A. P. J., and Peberdy, J. F. (1999) Glucoamylase overexpression and secretion in Aspergillus niger: analysis of glycosylation, BBA-Gen. Subjects 1472, 576–86.
Medina, M. L., Haynes, P. A., Breci, L., and Francisco, W. A. (2005) Analysis of secreted proteins from Aspergillus flavus, Proteomics 5, 3153–61.
Medina, M. L., Kiernan, U. A., and Francisco, W. A. (2004) Proteomic analysis of rutin-induced secreted proteins from Aspergillus flavus, Fungal. Genet. Biol. 41, 327–335.
Zhu, L. Y., Nguyen, C. H., Sato, T., and Takeuchi, M. (2004) Analysis of secreted proteins during conidial germination of Aspergillus oryzae RIB40, Biosci. Biotech. Bioch. 68, 2607–12.
Phalip, V., Delalande, F., Carapito, C., et al. (2005) Diversity of the exoproteome of Fusarium graminearum grown on plant cell wall, Curr. Genet. 48, 366–79.
Zorn, H., Peters, T., Nimtz, M., and Berger, R. G. (2005) The secretome of Pleurotus sapidus, Proteomics 5, 4832–38.
Keller, N. P. and Hohn, T. M. (1997) Metabolic pathway gene clusters in filamentous fungi, Fungal. Genet. Biol. 21, 17–29.
Semova, N., Storms, R., John, T., et al. (2006) Generation, annotation, and analysis of an extensive Aspergillus niger EST collection, BMC Microbiol. 6:7.
Fryksdale, B. G., Jedrzejewski, P. T., Wong, D. L., Gaertner, A. L., and Miller, B. S. (2002) Impact of deglycosylation methods on two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry for proteomic analysis, Electrophoresis 23, 2184–93.
Edge, A. S. B., Faltynek, C. R., Hof, L., Reichert, L. E., and Weber, P. (1981) Deglycosylation of glycoproteins by trifluoromethanesulfonic acid, Anal. Biochem. 118, 131–7.
Suarez, M. B., Sanz, L., Chamorro, M. I., et al. (2005) Proteomic analysis of secreted proteins from Trichoderma harzianum - Identification of a fungal cell wall-induced aspartic protease, Fungal Genet. Biol. 42, 924–34.
Bradford, M. M. (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding, Anal. Biochem. 72, 248–54.
Nandakumar, M. P., Shen, J., Raman, B., and Marten, M. R. (2003) Solubilization of trichloroacetic acid (TCA) precipitated microbial proteins via NaOH for hboxtwo-dimensional electrophoresis, J. Proteome Res. 2, 89–93.
Acknowledgments
The techniques described here were adapted from the appropriate original papers. This work was supported by NSF grant MCB-0317126 to W.A.F. M.L.M. was supported in part by a fellowship through the Research Training Group in Optical Biomolecular Devices provided under NSF grant DBI-9602258-003 and the NSF-funded MGE@MSA Program, an Alliance for Graduate Education and the Professoriate, headquartered at Arizona State University.
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Medina, M.L., Francisco, W.A. (2008). Isolation and Enrichment of Secreted Proteins from Filamentous Fungi. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 425. Humana Press. https://doi.org/10.1007/978-1-60327-210-0_22
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DOI: https://doi.org/10.1007/978-1-60327-210-0_22
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