Purification and characterization of a membrane glycoprotein from the fish pathogen Flavobacterium psychrophilum.
OBJECTIVE
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.


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.


CONCLUSIONS
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.
Merle
C
C
Laboratoire de Microbiologie et de Biochimie Appliquées, Ecole Nationale d'Ingénieurs des Travaux Agricoles de Bordeaux, Gradignan Cedex, France.
Faure
D
D
Urdaci
M-C
MC
Le Hénaff
M
M
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005393:Fish Diseases / Q000382:microbiology*; D005417:Flavobacterium / Q000378:metabolism*; D015151:Immunoblotting / Q000379:methods; D007122:Immunoelectrophoresis / Q000379:methods; D008562:Membrane Glycoproteins / Q000302:isolation & purification*; D012487:Salmonidae / Q000382:microbiology*
D008562:Membrane Glycoproteins