High-performance liquid chromatographic separation of Porphyromonas gingivalis fimbriae

Abstract

Fimbriae are responsible for the adherence of many bacterial species to the surfaces they eventually colonize. Porphyromonas gingivalis is an important pathogenic agent involved in periodontal disease. Fimbriae of P. gingivalis have been thought to mediate binding of the bacterium to oral surfaces. In order to study the role of fimbriae in microbial adhesion, it is important to purify fimbriae to homogeneity. A simple and rapid reverse-phase high-performance liquid chromatography (HPLC) method is developed to purify P. gingivalis fimbriae. The crude fimbriae were precipitated from sonic extract of P. gingivalis cells with the 40% ammonium sulfate precipitation. The dialyzed crude fimbriae preparation was subjected to reverse-phase HPLC separation. The purity and size of purified fimbrial proteins were confirmed by SDS-polyacrylamide gel electrophoresis(SDS-PAGE) followed by Western immunoblot using polyclonal antibodies against fimbriae. The purified major fimbrial protein from strain 33277 of P. gingivalis had apparent molecular mass of 41 kDa. The method is useful for analytical as well as preparative purification with 25% yields from the ammonium sulfate-precipitated crude fimbriae preparation, and represents increased speed and efficiency over earlier published methods.