The molecular cloning, nucleotide sequence and expression of an antigenic determinant from Porphyromonas gingivalis.
A genomic library generated in Escherichia coli was probed with a monoclonal antibody (mAb) LDS28, which reacts with a species-specific cell-surface antigen of Porphyromonas gingivalis. A clone designated pGPR2.1 was shown to express a 46-kDa protein reactive with mAb LDS28, which maps to a 1.7-kb HincII fragment. DNA sequence analysis revealed pGPR2.1 contains a 5653-bp insert with six open reading frames, one of which shows significant DNA homology with the rnhB gene of E. coli. Several subclones of pGPR2.1 were randomly generated in plasmid vector pTTQ18* using restriction enzyme Sau3a. Immunoblotting of subclones demonstrated that the LDS28-reactive antigen was coded for by an open reading frame predicted to specify a protein of 455 amino acids (50 kDa). This open reading frame was designated pgaA (Porphyromonas gingivalis antigen). The predicted amino acid sequence of PgaA contains a putative ABC signature for binding NTPs as well as a predicted transmembrane domain. Minicell labelling of pGPR2.1-encoded proteins and subclone derivatives revealed that pgaA directs expression of protein of multiple molecular weights (31-46 kDa) from its own promoter in E. coli, and that some of these forms may be caused by proteolysis of a 50-kDa precursor which itself shows a reduced apparent molecular weight (46 kDa) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis.
Rigg
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GP
Department of Pathological Sciences, University of Manchester, Manchester Royal Infirmary, UK.
Roberts
I S
IS
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016966:Porphyromonas gingivalis / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013462:Sulfur Radioisotopes
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000954:Antigens, Surface; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; C404486:pgaA protein, Porphyromonas gingivalis; D008715:Methionine