Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi

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• 作者：Daniela Isola^a ; ^{isola@unitus.it} ; Gorji Marzban^b ; ^{gorji.marzban@boku.ac.at} ; Laura Selbmann^a ; ^{selbmann@unitus.it} ; Silvano Onofri^a ; ^{onofri@unitus.it} ; Margit Laimer^b ; ^{margit.laimer@boku.ac.at} ; Katja Sterflinger^c ; ^{katja.sterflinger@boku.ac.at}
• 关键词：Black meristematic fungi ; Black yeast ; Protein extraction method ; Rock inhabiting fungi
• 刊名：Fungal Biology
• 出版年：2011
• 出版时间：October 2011
• 年：2011
• 卷：115
• 期：10
• 页码：971-977
• 全文大小：670 K

文摘

The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction?was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium?perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.