title: Expression of Porphyromonas gingivalis proteolytic activity in Escherichia coli.
abstract: Porphyromonas gingivalis (formerly Bacteroides gingivalis) degrades numerous protein substrates including collagen, fibrinogen, fibronectin, gelatin, casein, immunoglobulins and complement components. In order to clone one or more of these protease genes, a genomic library was constructed with Sau3A1 restriction fragments of chromosomal DNA from P. gingivalis ATCC 33277 ligated into the temperature-regulated vector pCQV2, and expressed in Escherichia coli DH5 alpha mcr. The electro-transformants (3 x 10(4)) were screened for general protease activity on Luria broth agar containing ampicillin (50 mg/l) and sodium caseinate (2%). One casein-hydrolyzing clone was detected and subcultured, and the activity of the cell extracts was characterized. We were able to show that the protease-positive clone, (pTEM1), had broad substrate specificity. Colorimetric assays indicated the hydrolysis of azocoll, azocasein, collagen, elastin-congo red and artificial substrates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to confirm that collagen, casein, fibrinogen and fibronectin were degraded by the clone.
authors.affiliation: Department of Dental Research, University of Rochester, New York.
authors.forename: T E
authors.initials: TE
authors.lastname: Madden
authors.forename: T M
authors.initials: TM
authors.lastname: Thompson
authors.forename: V L
authors.initials: VL
authors.lastname: Clark
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D002364:Caseins / Q000378:metabolism; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D016966:Porphyromonas gingivalis / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002364:Caseins; D005353:Fibronectins; D005340:Fibrinogen; D003094:Collagen; D010450:Endopeptidases