title: Generation and purification of recombinant fimbrillin from Porphyromonas (Bacteroides) gingivalis 381.
abstract: Fimbrillin is the major subunit protein of fimbriae from the human periodontal pathogen Porphyromonas (Bacteroides) gingivalis. We describe here the generation and initial characterization of recombinant fimbrillin (r-fimbrillin) isolated from P. gingivalis 381. A fragment of DNA encoding the gene for fimbrillin was generated by polymerase chain reaction and cloned into the expression vector pET11b. Plasmids containing the recombinant gene were transfected into Escherichia coli. Clones were selected on plates for ampicillin resistance and individually screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for protein production after activation with IPTG (isopropyl-beta-D- thiogalactopyranoside). One clone, OW0.2, produced significant amounts of a 42-kDa protein after induction with IPTG. This clone contained the pET11b plasmid with a 1-kb insert that had sequence homology to the gene encoding fimbrillin. The majority of recombinant protein from clone OW0.2 was found in the cytoplasm within inclusion bodies. Protein aggregates were solubilized in 8 M urea, and SDS-PAGE analysis showed two major protein bands, one at 42 kDa and the other at 17 kDa. These two proteins coeluted from a DEAE-Sepharose column at 0.15 M NaCl and were reactive to rabbit antiserum to fimbrillin in a Western blot (immunoblot). A preparation giving a single protein band at 42 kDa in SDS-PAGE was obtained by size fractionation by using continuous-elution electrophoresis. Lymph node cells from animals immunized with either fimbrillin from P. gingivalis or r-fimbrillin showed antigen-specific proliferation to both P. gingivalis fimbrillin and r-fimbrillin in an in vitro recall assay. Therefore, it appears that r-fimbrillin is chemically, antigenically, and serologically identical to fimbrillin isolated from P. gingivalis 381.
authors.affiliation: Department of Periodontics, University of North Carolina, Chapel Hill 27599.
authors.forename: O R
authors.initials: OR
authors.lastname: Washington
authors.forename: M
authors.initials: M
authors.lastname: Deslauriers
authors.forename: D P
authors.initials: DP
authors.lastname: Stevens
authors.forename: L K
authors.initials: LK
authors.lastname: Lyford
authors.forename: S
authors.initials: S
authors.lastname: Haque
authors.forename: Y
authors.initials: Y
authors.lastname: Yan
authors.forename: P M
authors.initials: PM
authors.lastname: Flood
mesh_terms: D000818:Animals; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D005260:Female; D036781:Fimbriae Proteins; D010861:Fimbriae, Bacterial / Q000276:immunology*; D008213:Lymphocyte Activation; D051379:Mice; D008808:Mice, Inbred CBA; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016966:Porphyromonas gingivalis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000302:isolation & purification
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C063055:fimbrillin; D036781:Fimbriae Proteins