Purification, characterization, and localization of a major membrane protein antigen from Porphyromonas (bacteroides) gingivalis.
Humans and rats infected with P. gingivalis develop a strong immune response to a 75 kDa major membrane component of P. gingivalis and hence knowledge of the nature of this molecule may aid in understanding the host response to P. gingivalis during infection. Purification of the 75 kDa protein was achieved by repeated precipitation from a crude sonicate of P. gingivalis 2561 at pH 5.0. Homogeneity of the purified 75 kDa protein was confirmed by SDS-PAGE and Western immunoblot analysis using monoclonal and polyclonal antibodies. The purified protein revealed an apparent molecular mass of 300 kDa in native form. Although most of the strains of P. gingivalis tested showed a major membrane protein band in the range of 61 to 78 kDa, on Western immunoblot analysis only the strains which have proteins in the range of 75 to 78 kDa were reactive with anti-75 kDa protein polyclonal antibodies. Affinity purified polyclonal antibodies were used to localized 75 kDa protein on the cell surface of P. gingivalis 2561 by immunogold electron microscopy. Immunolabeling of the 75 kDa protein demonstrated specific localization of the protein along the outer cell membrane, but not on the fimbriae. Furthermore, immunogold labeling of the 75 kDa protein on the thin sections showed that the 75 kDa component was present on not only the outer membrane, but also on the cell membrane, and on membrane bound organelles. Localization of this protein suggests that the 75 kDa component is a membrane-associated protein.
Sojar
H T
HT
State University of New York, Department of Oral Biology, Buffalo 14214.
Lee
J Y
JY
Bedi
G S
GS
Cho
M I
MI
Genco
R J
RJ
D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D000940:Antigenic Variation / Q000276:immunology; D000942:Antigens, Bacterial / Q000302:isolation & purification*; D001425:Bacterial Outer Membrane Proteins / Q000276:immunology*; D001439:Bacteroides / Q000276:immunology*; D015153:Blotting, Western; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008970:Molecular Weight
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins