Isolation and Characterization of Major Outer Membrane Proteins of Edwardsiella tarda.

Abstract

Three different extraction methods were used for preparing major outer membrane protein (MOMP) from the cell membrane fraction ofEdwardsiella tarda EF-1 which was isolated from Japanese eelAnguilla japonica. Triton X-100 extracted many proteins, including a thick band at 97 kDa in SDS-PAGE. Sodium dodecyl sulfate (SDS) and sodium lauryl sarcosinate (Sarkosyl), however, extracted three MOMPs at 37, 40 and 43 kDa, suggesting that these three MOMPs are closely associated to the membrane. The mobilities of the 37, 40 and 43 kDa MOMPs in SDS-PAGE were not affected by 2-mercaptoethanol treatment. Native-PAGE of the SDS-extracted MOMPs showed an unseparated protein band only at high molecular weight range. These findings indicate that the three MOMPs compose large molecules without intramolecular disulfide bridges in the outer membrane ofE. tarda EF-1. These MOMPs were distinctly detected in the western blotting with rabbit antiserum against EF-1 whole cell, indicating the three MOMPs are involved in the determination of the cell antigenicity. SDS-PAGE profiles of the MOMPs prepared by SDS-method from 15E tardastrains, which were isolated from Japanese eel, yellowtail Seriola quinqueradiata, Japanese flounderParalichthys olivaceusand crimson sea breamEvynnis japonicain Japan, showed a major protein located at 37 kDa, although 2 strains from Taiwan showed a slightly smaller molecular weight size. This MOMP was visualized in western blotting by using the anti-Etarda EF-1 serum, with no cross reaction of MOMPs of bacterial species in other genera :Escherichia, Photobacterium, Pseudomonas, AeromonasandVibrio. The results indicate an epitope included in the MOMP located around 37 kDa is common amongE. tardastrains and specific to this species.