Purification of major fimbrial proteins of Porphyromonas gingivalis.
Binding of Porphyromonas gingivalis to pellicle-coated teeth is mediated to a large extent by fimbrillin, a major structural subunit of fimbriae. A simple and efficient method for purifying the major fimbrial proteins from various strains of P. gingivalis was developed. The sonic extracts of crude fimbriae were prepared from P. gingivalis strains 2561, A7A1-28, EM-3, and 9-14K-1, which are representatives of different fimbrial groups. The crude fimbriae were precipitated from the extracts with 40% saturated ammonium sulfate. The dialyzed crude fimbriae were dissolved in 8 M guanidine HCl and loaded onto a Sepharose CL-6B column equilibrated with 6 M guanidine HCl. The major fimbrial protein-enriched fractions were obtained and further purified to homogeneity by repetitive gel filtration on the same column. The purity and intactness of the purified fimbrial proteins were ascertained by SDS-polyacrylamide gel electrophoresis followed by silver nitrate staining and immunoblot analysis using rabbit antisera raised against the purified fimbrial proteins. The result demonstrates the usefulness of guanidine HCl as a reliable reagent for purifying various fimbrial proteins of P. gingivalis, which are often associated strongly with other outer membrane proteins and show different physicochemical and antigenic characteristics.
Lee
J Y
JY
Department of Oral Microbiology, School of Dentistry, Kyung Hee University, Seoul, Korea.
Sojar
H T
HT
Amano
A
A
Genco
R J
RJ
D000645:Ammonium Sulfate; D000818:Animals; D000907:Antibodies, Bacterial; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000737:chemistry* / Q000276:immunology; D019791:Guanidine; D006146:Guanidines; D006801:Humans; D007202:Indicators and Reagents; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000276:immunology; D011817:Rabbits
D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D006146:Guanidines; D007202:Indicators and Reagents; C063055:fimbrillin; D036781:Fimbriae Proteins; D019791:Guanidine; D000645:Ammonium Sulfate