Expression of Porphyromonas gingivalis proteolytic activity in Escherichia coli.
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.
Madden
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TE
Department of Dental Research, University of Rochester, New York.
Thompson
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TM
Clark
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VL
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
D001426:Bacterial Proteins; D002364:Caseins; D005353:Fibronectins; D005340:Fibrinogen; D003094:Collagen; D010450:Endopeptidases