Rathore, Dharmendar ; Batra, Janendra K. (1997) Construction, expression and characterization of chimaeric toxins containing the ribonucleolytic toxin restrictocin: intracellular mechanism of action Biochemical Journal, 324 . pp. 815-822. ISSN 0264-6021
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Official URL: http://www.biochemj.org/bj/324/bj3240815.htm
Abstract
Restrictocin is a ribonucleolytic toxin produced by the fungus Aspergillus restrictus. Two chimaeric toxins containing restrictocin directed at the human transferrin receptor have been constructed. Anti-TFR(scFv)-restrictocin is encoded by a gene produced by fusing the DNA encoding a single-chain antigen-combining region (scFv) of a monoclonal antibody, directed at the human transferrin receptor, at the 5' end of that encoding restrictocin. The other chimaeric toxin, restrictocin-anti-TFR(scFv), is encoded by a gene fusion containing the DNA encoding the single-chain antigen-combining region of antibody to human transferrin receptor at the 3' end of the DNA encoding restrictocin. These gene fusions were expressed in Escherichia coli, and fusion proteins purified from the inclusion bodies by simple chromatography techniques to near-homogeneity. The two chimaeric toxins were found to be equally active in inhibiting protein synthesis in a cell-free in vitro translation assay system. The chimaeric toxins were selectively toxic to the target cells in culture with potent cytotoxic activities. However, restrictocin-anti-TFR(scFv) was more active than anti-TFR(scFv)-restrictocin on all cell lines studied. By using protease and metabolic inhibitors, it can be shown that, to manifest their cytotoxic activity, the restrictocin-containing chimaeric toxins need to be proteolytically processed intracellularly and the free toxin or a fragment thereof thus generated is translocated to the target via a route involving the Golgi apparatus.
Item Type: | Article |
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Source: | Copyright of this article belongs to Portland Press Limited. |
ID Code: | 13346 |
Deposited On: | 11 Nov 2010 08:15 |
Last Modified: | 16 May 2016 22:32 |
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