Characterization of the Gene for the Hemin-Binding Protein from Porphyromonas Gingivalis.
10.5051/jkape.1999.29.3.663
- Author:
Sung Jo KIM
1
Author Information
1. Department of Periodontology, College of Dentistry, Pusan National University, Korea.
- Publication Type:Original Article
- Keywords:
hemin;
hemin-binding protein;
Porphyromonas gingivalis;
gene cloning
- MeSH:
Agar;
Clone Cells;
DNA;
Escherichia coli;
Genomic Library;
Hemin;
Humans;
Iron;
Periodontal Diseases;
Phenotype;
Plasmids;
Porphyromonas gingivalis*;
Porphyromonas*;
RNA
- From:The Journal of the Korean Academy of Periodontology
1999;29(3):663-675
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain DH5alpha, and screened for recombinant clones with heminbinding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa heminbinding protein.
