Purification of a Hemagglutinin Isolated from Non-Fimbriated Prevotella intermedia

Abstract

Previous studies have shown that amorphous structures, which are glycoprotein in nature, surrounding vesicle-like structures in non-fimbriated Prevotella intermedia strain E18 may mediate hemagglutination. We attempted to purify a hemagglutinin from Prevotella intermedia strain E18 cells by mechanical shearing, 30-50% ammonium sulfate precipitation, 10-60% sucrose density ultracentrifugation at 35,000rpm for 20h, Arginine Sepharose 4B column chromatography, and gel filtration.
      The active fraction was eluted from an Arginine Sepharose 4B column using 1M arginine. Activity was found at the second peak on a Sepharose CL-4B column when equilibrated with 10mM tris-HC1. However, the activity at the second peak was almost lost when equilibrated with 10mM tris-HC1 containing 6M urea as a starting buffer. These purification steps resulted in an increase of specific activity from 21,400 to 118,000AU/mg. In total, the purification procedure increased the specific activity 5.5-fold that of fraction A. A single band at approximately 25kDa was obtained by SDS-PAGE. Western blotting produced single bands corresponding to those from SDS-PAGE. Protein A-gold labeling of Prevotella intermedia strain E18 cells with anti-hemagglutinin IgG revealed that the IgG bound specifically to hemagglutinin that was present on the Prevotella intermedia strain E18 cell surface. These results indicate that this protein may be a hemagglutinin.