Preparation of intracellular proteins from a white-rot fungus surrounded by polysaccharide sheath and optimization of their two-dimensional electrophoresis for proteomic studies

- Ceriporiopsis subvermispora produced sheath in the presence of vanillin.
- We developed a rapid method to remove adherent sheath from C. subvermispora.
- The intracellular proteins were prepared from this fungus surrounded by sheath.
- By optimizing protein solubilization, we successfully separated these proteins.
- We applied the same proteins to two-dimensional difference gel electrophoresis.

The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound. Using this approach, we achieved thorough removal of sheath and cell disruption using beads and a solution with a high protein-solubilizing power, which enabled the efficient extraction of intracellular proteins from C. subvermispora surrounded by sheath. In addition, for proteomic analysis, we investigated whether these extracted proteins were compatible with two-dimensional electrophoresis. By efficiently concentrating on protein solubilization in the first dimension and using a stacking gel in the second dimension, we successfully obtained a high-resolution proteome map of C. subvermispora. We also used the same proteins for fluorescence two-dimensional difference gel electrophoresis to obtain the quantitative protein expression profiles. These steps demonstrated that two-dimensional electrophoresis-based proteomics can be used to clarify the composition of intracellular proteins from sheath-producing white-rot fungi.

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