A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia. Tavares, F. & Sellstedt, A. Journal of Microbiological Methods, 39(2):171–178, January, 2000. ![A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia [link]](https://bibbase.org/img/filetypes/link.svg "link")
Paper doi abstract bibtex A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris–HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.
@article{tavares_simple_2000,
title = {A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from {Frankia}},
volume = {39},
issn = {0167-7012},
url = {https://www.sciencedirect.com/science/article/pii/S0167701299001153},
doi = {10.1016/S0167-7012(99)00115-3},
abstract = {A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris–HCl (pH 6.8) buffer supplemented with 0.1\% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51\%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.},
language = {en},
number = {2},
urldate = {2021-11-08},
journal = {Journal of Microbiological Methods},
author = {Tavares, Fernando and Sellstedt, Anita},
month = jan,
year = {2000},
keywords = {Actinomycetes, Cell fractionation, Gram-positive cell wall, Peptidoglycan hydrolases},
pages = {171--178},
}
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Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. 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The method was based on washing Frankia mycelia in 62.5 mM Tris–HCl (pH 6.8) buffer supplemented with 0.1\\% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5±7.44 μg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50±0.51\\%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. 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