Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum |
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Authors: | Merle C Faure D Urdaci M-C Le Hénaff M |
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Institution: | Laboratoire de Microbiologie et de Biochimie Appliquées, Ecole Nationale d'Ingénieurs des Travaux Agricoles de Bordeaux, Gradignan Cedex, France. |
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Abstract: | AIMS: The cell envelope of the fish pathogen Flavobacterium psychrophilum contains more than 50 polypeptides resolved by sodium dodecyl sulphate-polyacrylaminde gel electrophoresis analysis including a major component named P60. Here, we have developed a simple and efficient procedure for the purification of P60 and therefore permitting its biochemical characterization. METHODS AND RESULTS: Membrane proteins were selectively extracted from isolated cell envelopes with the mild non-ionic detergent Triton X-100. About 10 polypeptides were identified from the detergent fraction, including P60. The P60-enriched fraction was thereafter subjected to an anion exchange chromatographic step in the presence of Triton X-100. The molecule was purified at the milligram level (yield, about 75%; purification factor, 6.2). Analyses performed by charge shift electrophoresis, Triton X-114 phase separation and by detection of sugar-modified components showed that P60 is a true amphiphilic membrane-associated glycoprotein. CONCLUSIONS: The method described in this paper provides pure and non-denaturated P60 and should prove to be easily scaled-up. As sugar-modified protein, P60 should be included in the growing list of glycosylated prokaryotic proteins. SIGNIFICANCE AND IMPACT OF THE STUDY: It offers the possibility of obtaining P60 in amounts allowing the testing of the potential of P60 as a candidate for anti-flavobacteria subunit vaccines, as P60 is one of the major antigens. |
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Keywords: | Flavobacterium glycoproteins membrane proteins |
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