Co-expression of CdtA and CdtC subunits of cytolethal distending toxin from Aggregatibacter actinomycetemcomitans / 대한치주과학회지
The Journal of the Korean Academy of Periodontology
;
: 231-237, 2009.
Article
in English
| WPRIM
| ID: wpr-196937
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.
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Oligopeptides
/
Solubility
/
Bacterial Toxins
/
Enzyme-Linked Immunosorbent Assay
/
Glycolipids
/
Proteins
/
Edetic Acid
/
Cytotoxins
/
Escherichia coli
/
Histidine
Limits:
Humans
Language:
English
Journal:
The Journal of the Korean Academy of Periodontology
Year:
2009
Type:
Article
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