A study of a membrane-associated protease of porphyromonas gigngivalis W83
| Autor: | Park, Yoonsuk |
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| Jazyk: | angličtina |
| Rok vydání: | 1995 |
| Druh dokumentu: | Text |
| Popis: | A protease gene (tpr) of Porphyromonas gingivalis W83 was cloned and expressed in Escherichia coli. The recombinant protease with an apparent molecular mass of 90 kDa was detected as a proteolytic band on bovine serum albumin (BSA)-substrate zymogram and characterized as a thiol-dependent protease. Western immunoblot analysis and in-vitro translation showed that the tpr gene encodes a 50-kDa polypeptide, suggesting that the 90- kDa active protease may be composed of a subunit having a molecular mass 50 kDa. The recombinant protein was expressed at high levels using a T7 RNA polymerase/promoter system. Antiserum raised to the recombinant protein was reactive to a native P. gingivalis protein with a molecular mass of 80 kDa. A specific protease (Tpr)-deficient isogenic mutant, P. gingivalis W83/PM, was generated by homologous recombination between P. gingivalis W83 chromosomal DNA and a suicide plasmid carrying the inactivated tpr gene in which a portion of the gene was replaced with an erythromycin resistance gene. Gelatin-substrate zymography and Western immunoblot analysis showed that W83/PM did not express the 80-kDa protein seen in the parent strain. Compared with W83, the mutant W83/PM showed a greatly reduced ability to hydrolyze the bacterial collagenase substrate, p-phenylazobenzyloxycarbonyl-L-prolyl-leucyl-glycyl-Lprolyl- D-arginine (Pz-peptide). The enzyme is associated with the cell membrane of W83. The Pz-peptidase activity of P. gingivalis W83 was characterized. No significant differences were found in the ability of W83 or W83/PM to hydrolyze collagen, or in their pathogenicity in a mouse model. To determine the role of Tpr in nutrient uptake by P. gingivalis, W83 and W83/PM were cultivated in media containing limiting amounts of amino acid/peptide nutrients and in the growth limiting media supplemented with BSA or gelatin. The growth of both strains was enhanced by BSA or gelatin. Cells of W83 grown in the nutrient limited media expressed higher levels of Tpr. A complementation study was performed by introducing the recombinant shuttle plasmid pBY3 containing the tpr gene into W83/PM. Expression of Tpr by the complemented strain was observed by gelatin-substrate zymography, Western immunoblot analysis and Pz-peptidase assay. Science, Faculty of Microbiology and Immunology, Department of Graduate |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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