Cloning and protein expression of Aggregatibacter actinomycetemcomitans cytolethal distending toxin C
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Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease. (J Korean Acad Periodontol 2008;38:317-324)
Abstract
Purpose: Aggregatibacter actinomycetemcomitans was associated with localized aggressive periodontitis, endocarditis, meningitis, and osteomyelitis. The cytolethal distending toxin (CDT) of A. actinomycetemcomitans was considered as a key factor of these diseases is composed of five open reading frames (ORFs). Among of them, An enzymatic subunit of the CDT, CdtB has been known to be internalized into the host cell in order to induce its genotoxic effect. However, CdtB can not be localized in host cytoplasm without the help of a heterodimeric complex consisting of CdtA and CdtC. So, some studies suggested that CdtC functions as a ligand to interact with GM3 ganglioside of host cell surface. The precise role of the CdtC protein in the mechanism of action of the holotoxin is unknown at the present time. The aim of this study was to generate recombinant CdtC proteins expression from A. actinomycetemcomitans, through gene cloning and protein used to investigate the function of Cdt C protein in the bacterial pathogenesis Materials and Methods: The genomic DNA of A. actinomycetemcomitans Y4 (ATCC29522) was isolated using the genomic DNA extraction kit and used as template to yield cdtC genes by PCR. The amplifed cdtC genes were cloned into T-vector and cloned cdt C gene was then subcloned to pET28a expression vector. The pET28a-cdtC plasmid expressed in BL21 (DE3) Escherichia coli system. Diverse conditons were tested to opitimize the expression and purification of functional CdtC protein in E. coli. Results: In this study we reconstructed CdtC subunit of A. actinomycetemcomitans Y4 and comfirmed the recombinant CdtC expression by SDS-PAGE and Western Blotting. The expression level of the recombinant CdtC was about 2% of total bacterial proteins. Conclusion: The lab condition of procedure for the purification of functionally active recombinant CdtC protein is established. The active recombinant CdtC protein will serve to examine the role of CdtC proteins in the host recognition and enzyme activity of CDT and investigate the pathological process of A. actinomycetemcomitans in periodontal disease. (J Korean Acad Periodontol 2008;38:317-324)
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