Characterization of the tpr gene product and isolation of a specific protease-deficient mutant of Porphyromonas gingivalis W83.
The previously described protease gene (tpr) of Porphyromonas gingivalis W83 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the recombinant protein and in vitro translation to encode a 50-kDa protein whose active form migrates with an apparent molecular mass of 90 kDa. The 50-kDa protein was expressed at high levels by using a T7 RNA polymerase/promoter system. The NH2-terminal sequence of the protein was identical to the amino acid sequence deduced from the DNA sequence of the protease gene. Affinity-purified antibody to the 90-kDa recombinant protease reacted with an 80-kDa P. gingivalis protein. A specific protease (Tpr)-deficient isogenic mutant of P. gingivalis was generated by homologous recombination between P. gingivalis chromosomal DNA and a suicide plasmid carrying the cloned gene disrupted by insertion of an erythromycin resistance gene. Gelatin substrate zymography showed that cell extracts of the mutant lacked a protease band that migrated with an apparent molecular mass of 80 kDa. Western immunoblots of the cell extracts indicated the loss of an antigen with a similar mass.
Park
Y
Y
Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
McBride
B C
BC
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010450:Endopeptidases