A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia

• Type: Article in International Scientific Journal
• Date: 2000

Authors

Tavares, F (Author) FCUP; Sellstedt, A (Author) Other

Journal

Title: Journal of Microbiological Methods

Vol. 39

Pages: 171-178

ISSN: 0167-7012

Publisher: Elsevier

Indexing

ISI Web of Knowledge - 21 Citations

Scopus - 24 Citations

Scientific classification

FOS: Natural sciences > Biological sciences

Other information

Authenticus ID: P-001-1SN

DOI: 10.1016/s0167-7012(99)00115-3

Abstract (EN): 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 pg 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: English

Type (Professor's evaluation): Scientific

No. of pages: 8