title: Synthesis and assembly of Porphyromonas gingivalis fimbrial protein in potato tissues.
abstract: Periodontal disease caused by the gram-negative oral anaerobic bacterium Porphyromonas gingivalis is thought to be initiated by the binding of P. gingivalis fimbrial protein to saliva-coated oral surfaces. To assess whether biologically active fimbrial antigen can be synthesized in edible plants, a cDNA fragment encoding the C-terminal binding portion of P. gingivalis fimbrial protein, fimA (amino acids 266-337), was cloned behind the mannopine synthase promoter in plant expression vector pPCV701. The plasmid was transferred into potato (Solanum tuberosum) leaf cells by Agrobacterium tumefaciens in vivo transformation methods. The fimA cDNA fragment was detected in transformed potato leaf genomic DNA by PCR amplification methods. Further, a novel immunoreactive protein band of ~6.5 kDa was detected in boiled transformed potato tuber extracts by acrylamide gel electrophoresis and immunoblot analysis methods using primary antibodies to fimbrillin, a monomeric P. gingivalis fimbrial subunit. Antibodies generated against native P. gingivalis fimbriae detected a dimeric form of bacterial-synthesized recombinant FimA(266-337) protein. Further, a protein band of ~160 kDa was recognized by anti-FimA antibodies in undenatured transformed tuber extracts, suggesting that oligomeric assembly of plant-synthesized FimA may occur in transformed plant cells. Based on immunoblot analysis, the maximum amount of FimA protein synthesized in transformed potato tuber tissues was approximately 0.03% of total soluble tuber protein. Biosynthesis of immunologically detectable FimA protein and assembly of fimbrial antigen subunits into oligomers in transformed potato tuber tissues demonstrate the feasibility of producing native FimA protein in edible plant cells for construction of plant-based oral subunit vaccines against periodontal disease caused by P. gingivalis.
authors.affiliation: Department of Biochemistry and Microbiology, Center for Health Disparities and Molecular Medicine, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA.
authors.forename: Eun-Ah
authors.initials: EA
authors.lastname: Shin
authors.forename: Yong Keun
authors.initials: YK
authors.lastname: Park
authors.forename: Kang Oh
authors.initials: KO
authors.lastname: Lee
authors.forename: William H R
authors.initials: WH
authors.lastname: Langridge
authors.forename: Jin-Yong
authors.initials: JY
authors.lastname: Lee
mesh_terms: D036781:Fimbriae Proteins / Q000096:biosynthesis* / Q000502:physiology; D005526:Food, Formulated; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D011198:Solanum tuberosum / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; C063055:fimbrillin; D036781:Fimbriae Proteins