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Generation of a recombinant Aggregatibacter actinomycetemcomitans RTX toxin in Escherichia coli.
- Source :
-
Gene [Gene] 2018 Sep 25; Vol. 672, pp. 106-114. Date of Electronic Publication: 2018 Jun 04. - Publication Year :
- 2018
-
Abstract
- A leukotoxin (LtxA) that is produced by Aggregatibacter actinomycetemcomitans (Aa) is an important virulence determinant in an aggressive form of periodontitis in adolescents. Understanding the function of this protein at the molecular level is critical to elucidating its role in the disease process. To accomplish genetic analysis of the protein structure and relating these observations to toxin function, we have developed an E. coli expression system for the generation and rapid purification of LtxA. Cloning the structural toxin gene, ltxA, from Aa strain JP2 under control of T7 promoter-1 of pCDFDuet-1 vector resulted in expression of a 114 KDa protein which could be easily purified by the presence of a carboxy-terminal engineered double hexahistidine (double-His <subscript>6</subscript> ) tag and was immunologically reactive with an anti-LtxA monoclonal antibody, but was not cytotoxic. Cloning a second gene, ltxC, an acyltransferase gene, into the vector under control of T7 promoter-2, resulted in expression of the biologically active LtxA. The toxin was extracted from E. coli inclusion bodies, purified by immobilized metal affinity chromatography, and refolded by dialysis. When compared by circular dichroism (CD) spectroscopy analysis, acylated recombinant LtxA has a secondary structure consistent with wt LtxA, while variations in α-helical structure of nonacylated LtxA were observed. No modifications in α-helix were found upon the toxin's binding with liposome-incorporated cholesterol. Our results suggest that pure, biologically active recombinant LtxA can be isolated by a one-step affinity chromatography from E. coli. The toxic and structural properties of the recombinant LtxA are similar to its wt counterpart.<br /> (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Subjects :
- Bacterial Toxins biosynthesis
Bacterial Toxins chemistry
Bacterial Toxins isolation & purification
Cloning, Molecular
Escherichia coli genetics
Escherichia coli growth & development
Escherichia coli metabolism
Exotoxins biosynthesis
Exotoxins chemistry
Exotoxins isolation & purification
Humans
Protein Structure, Secondary
Recombinant Proteins biosynthesis
Recombinant Proteins genetics
THP-1 Cells
Aggregatibacter actinomycetemcomitans genetics
Bacterial Toxins genetics
Exotoxins genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1879-0038
- Volume :
- 672
- Database :
- MEDLINE
- Journal :
- Gene
- Publication Type :
- Academic Journal
- Accession number :
- 29879499
- Full Text :
- https://doi.org/10.1016/j.gene.2018.06.003