Cloning of aPorphyromonas (Bacteroides) gingivalisprotease gene and characterization of its product

Autor: Yoonsuk Park, Barry C. McBride
Rok vydání: 1992
Předmět:
Zdroj: FEMS Microbiology Letters. 92:273-278
ISSN: 1574-6968
0378-1097
DOI: 10.1111/j.1574-6968.1992.tb05273.x
Popis: A clone expressing a Porphyromonas gingivalis protease from the recombinant plasmid (pYS307) has been identified in a genomic library of P. gingivalis W83. The cloned gene was localized to a 2.4-kb DNA fragment between Bam HI and Hind III sites. When a 3.2-kb Hin dIII fragment of pYS307 was used as a probe in Southern hybridization, Hin dIII-digested chromosomal DNA of P. gingivalis W83, as well as those of W50 and W12, showed a single 3.2-kb hybridizing band, while that of P. gingivalis 33277 showed a 5.0-kb band. Colonies of E. coli containing pYS307 showed pronounced proteolytic zones on skim milk agar plates only when incubated in an oxygen-free environment. BSA substrate zymography of whole cell extract of E. coli containing pYS307 revealed a protease of approx. 80 kDa which was active under reducing conditions. These results suggest that the cloned protease is thiol-dependent. Antiserum to P. gingivalis W50 reacted with a single band of 80 kDa when a cell lysate sample of an E. coli JM83 containing pYS307 was prepared for electrophoresis in the absence of β-mercaptoethanol. When samples were solubilized in the presence of β-mercaptoethanol prior to electrophoresis, the antiserum reacted with the bands of 50 and 38 kDa, but there was no reaction observed at 80 kDa. The activity of the cloned protease was inhibited by TLCK, TPCK, EDTA, PMSF, iodoacetic acid and ZnCl2.
Databáze: OpenAIRE
Externí odkaz: