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. 1993 Aug;61(8):3570–3573. doi: 10.1128/iai.61.8.3570-3573.1993

Expression of a functional Porphyromonas gingivalis fimbrillin polypeptide in Escherichia coli: purification, physicochemical and immunochemical characterization, and binding characteristics.

A Sharma 1, H T Sojar 1, J Y Lee 1, R J Genco 1
PMCID: PMC281044  PMID: 8392975

Abstract

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.

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Selected References

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  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Boyd J., McBride B. C. Fractionation of hemagglutinating and bacterial binding adhesins of Bacteroides gingivalis. Infect Immun. 1984 Aug;45(2):403–409. doi: 10.1128/iai.45.2.403-409.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dickinson D. P., Kubiniec M. A., Yoshimura F., Genco R. J. Molecular cloning and sequencing of the gene encoding the fimbrial subunit protein of Bacteroides gingivalis. J Bacteriol. 1988 Apr;170(4):1658–1665. doi: 10.1128/jb.170.4.1658-1665.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Evans R. T., Klausen B., Sojar H. T., Bedi G. S., Sfintescu C., Ramamurthy N. S., Golub L. M., Genco R. J. Immunization with Porphyromonas (Bacteroides) gingivalis fimbriae protects against periodontal destruction. Infect Immun. 1992 Jul;60(7):2926–2935. doi: 10.1128/iai.60.7.2926-2935.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Goulbourne P. A., Ellen R. P. Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus. J Bacteriol. 1991 Sep;173(17):5266–5274. doi: 10.1128/jb.173.17.5266-5274.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Isogai H., Isogai E., Yoshimura F., Suzuki T., Kagota W., Takano K. Specific inhibition of adherence of an oral strain of Bacteroides gingivalis 381 to epithelial cells by monoclonal antibodies against the bacterial fimbriae. Arch Oral Biol. 1988;33(7):479–485. doi: 10.1016/0003-9969(88)90028-3. [DOI] [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Lee J. Y., Sojar H. T., Bedi G. S., Genco R. J. Porphyromonas (Bacteroides) gingivalis fimbrillin: size, amino-terminal sequence, and antigenic heterogeneity. Infect Immun. 1991 Jan;59(1):383–389. doi: 10.1128/iai.59.1.383-389.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lee J. Y., Sojar H. T., Bedi G. S., Genco R. J. Synthetic peptides analogous to the fimbrillin sequence inhibit adherence of Porphyromonas gingivalis. Infect Immun. 1992 Apr;60(4):1662–1670. doi: 10.1128/iai.60.4.1662-1670.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Naito Y., Gibbons R. J. Attachment of Bacteroides gingivalis to collagenous substrata. J Dent Res. 1988 Aug;67(8):1075–1080. doi: 10.1177/00220345880670080301. [DOI] [PubMed] [Google Scholar]
  11. Okuda K., Yamamoto A., Naito Y., Takazoe I., Slots J., Genco R. J. Purification and properties of hemagglutinin from culture supernatant of Bacteroides gingivalis. Infect Immun. 1986 Dec;54(3):659–665. doi: 10.1128/iai.54.3.659-665.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  13. Sojar H. T., Lee J. Y., Bedi G. S., Cho M. I., Genco R. J. Purification, characterization and immunolocalization of fimbrial protein from Porphyromonas (bacteroides) gingivalis. Biochem Biophys Res Commun. 1991 Mar 15;175(2):713–719. doi: 10.1016/0006-291x(91)91624-l. [DOI] [PubMed] [Google Scholar]
  14. Stinson M. W., Safulko K., Levine M. J. Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro. Infect Immun. 1991 Jan;59(1):102–108. doi: 10.1128/iai.59.1.102-108.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Washington O. R., Deslauriers M., Stevens D. P., Lyford L. K., Haque S., Yan Y., Flood P. M. Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381. Infect Immun. 1993 Mar;61(3):1040–1047. doi: 10.1128/iai.61.3.1040-1047.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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