Co-expression of CdtA and CdtC subunits of cytolethal distending toxin from Aggregatibacter actinomycetemcomitans.
10.5051/jkape.2009.39.S.231
- Author:
Seung Jae LEE
1
;
Kyung Yeol LEE
;
Hyung Seop KIM
Author Information
1. Department of Periodontology, College of Dentistry, Chonbuk National University, Korea. cbuperio@chonbuk.ac.kr
- Publication Type:Original Article
- Keywords:
Aggregatibacter actinomycetemcomitans;
toxin
- MeSH:
Antibodies;
Bacterial Toxins;
Cytotoxins;
Edetic Acid;
Enzyme-Linked Immunosorbent Assay;
Escherichia coli;
Glycolipids;
Histidine;
Humans;
Oligopeptides;
Proteins;
Solubility
- From:The Journal of the Korean Academy of Periodontology
2009;39(Suppl):231-237
- CountryRepublic of Korea
- Language:English
-
Abstract:
PURPOSE: Cytolethal distending toxin (CDT) is a family of heat-labile cytotoxins produced by several gram-negative mucosa-associated pathogens, including Aggregatibacter actinomycetemcomitans. CDT is well known to be capable of inducing growth arrest, morphological alterations, and eventually death in various cells. CDT belongs to a tripartite AB2 toxin (CdtB: the enzymatic A subunit ; CdtA and CdtC: the heterodimeric B subunit). Previous studies proposed that CdtA and CdtC together bind to a cell surface receptor and glycolipids act as a receptor for A. actinomycetemcomitans CDT (AaCDT). In this study, recombinant CdtA and CdtC proteins of AaCDT were co-expressed in a bacterial expression system and tested for their affinity for GM1 ganglioside. METHODS: The genes for CdtA and CdtC from A. actinomycetemcomitans Y4 were utilized to construct the expression vectors, pRSET-cdtA and pET28a-cdtC. Both CdtA and CdtC proteins were expressed in Escherichia coli BL21(DE3) and then purified using hexahistidine (His6) tag. The identity of purified protein was confirmed by anti-His6 antibody and monoclonal anti-CdtA antibody. Furthermore, the affinity of recombinant protein to GM1 ganglioside was checked through ELISA. RESULTS: Recombinant CdtA and CdtC proteins were expressed as soluble proteins and reacted to anti-His6 and monoclonal anti-CdtA antibodies. ELISA revealed that purified soluble CdtA-CdtC protein bound to GM1 ganglioside, while CdtA alone did not. CONCLUSIONS: Co-expression of CdtA and CdtC proteins enhanced the solubility of the proteins in E. coli, leading to convenient preparation of active CdtA-CdtC, a critical material for the study of AaCDT pathogenesis.
