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Separation and Comparison of Proteins From Virulent and Nonvirulent Strains of the Fish Pathogen Flavobacterium psychrophilum, Using a 2-D Electrophoretic Approach

e ability to separate cellular proteins based on their hydrophobicity was examined. The relationship of extracted proteins to cell surface proteins was then determined by comparison of 2-D gel profiles of biotin-labeled proteins. The results clearly demonstrate the ability of the extraction procedure to isolate proteins associated with cytoplasmic or cell membrane compartments of the bacteria (Figure 1).

Comparison of gel profiles shows that a large number of proteins are extracted in the E1 cell lysate fraction. Membrane-associated (less soluble) fractions were then obtained following E2 and E3 separation. Subsequent comparison to biotin-labeled proteins (Figure 2) shows that a dominant string of proteins (circled) at an approximate molecular weight of 100 kD and pI values between 5.2 and 5.8 represents a cell surface component and appears to primarily correspond to proteins in extraction E2 of Figure 1. Additional biotin-labeled proteins are present in the 20 kD range (pI approximately 5.15.6) but are not readily identified in the sequentially extracted fractions.

Comparison of Total Proteins from Virulent and Nonvirulent Strains of F. psychrophilum
Separation and comparison by 2-D PAGE of total proteins over the pH 310 range from strain CSF-259-93 (virulent) and ATCC 49418 (nonvirulent) demonstrate that proteins are differentially expressed between bacterial strains (Figure 3). Initial qualitative analysis reveals that the majority of bacterial proteins separate in the molecular weight ranges of approximately 40150 kD and have pI ranges of approximately 4.56.5. It appears that the nonvirulent ATCC strain has a higher concentration of proteins in this rang
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