J Korean Acad Periodontol.  2008 Aug;38(Suppl):343-354. 10.5051/jkape.2008.38.Suppl.343.

Purification and refolding of the recombinant subunit B protein of the Aggregatibacter actinomycetemcomitans cytolethal distending toxin

• Jeon YS
• Seo SC
• Kwon JH
• Ko SY
• Kim HS

• KMID: 1486869
• DOI: http://doi.org/10.5051/jkape.2008.38.Suppl.343

Abstract

PURPOSE
Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect.
MATERIALS AND METHODS
We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion.
RESULTS
The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions Mg2+ and Ca2+.
CONCLUSION
We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.

Keyword

Aggregatibacter actinomycetemcomitans; cytolethal distending toxin

MeSH Terms

Aggressive Periodontitis
Bacterial Toxins
Catalysis
Cell Cycle Checkpoints
Deoxyribonuclease I
Deoxyribonucleases
Dialysis
DNA
Edetic Acid
Escherichia coli
Humans
Inclusion Bodies
Ions
Meningitis
Osteomyelitis
Periodontitis
Plasmids
Polymerase Chain Reaction
Bacterial Toxins
DNA
Deoxyribonuclease I
Deoxyribonucleases
Edetic Acid
Ions