Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics.
Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. A structural subunit of the fimbriae, fimbrillin, has been shown to be important in binding of the bacterium to saliva-coated oral surfaces. In the present study, a coding region of the fimbrillin gene from P. gingivalis 2561 was amplified by the polymerase chain reaction and cloned into the pET-11d vector. The recombinant plasmid was transformed into Escherichia coli BL21, and protein expression was induced with isopropyl-beta-D-thiogalactopyranoside. The expressed protein was purified from insoluble inclusion bodies after solubilization with urea and gel filtration chromatography. The purified recombinant fimbrillin polypeptide, r-fim 10-337, corresponded to amino acid residues 10 to 337 of the deduced amino acid sequence of fimbrillin. In immunoblot analysis, r-fim 10-337 reacted with antibodies to fimbrillin purified from P. gingivalis, as well as with antibodies to synthetic peptides corresponding to the amino acid sequence of fimbrillin. The apparent molecular mass of r-fim 10-337 was estimated to be 41 kDa on sodium dodecyl sulfate-polyacrylamide gels. The r-fim 10-337 polypeptide was capable of inhibiting the binding of P. gingivalis 2561 to saliva-coated hydroxyapatite beads. These results suggest that the fimbrillin subunit polypeptide plays an important role in binding of P. gingivalis cells to saliva-coated surfaces. We describe here the successful expression and purification of a functionally and immunologically reactive recombinant P. gingivalis fimbrillin subunit from E. coli.
Sharma
A
A
Department of Oral Biology, State University of New York, Buffalo 14214.
Sojar
H T
HT
Lee
J Y
JY
Genco
R J
RJ
D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000276:immunology / Q000302:isolation & purification*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C063055:fimbrillin; D036781:Fimbriae Proteins