کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
1931160 | 1050543 | 2011 | 5 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Adaptation of Clostridium difficile toxin A for use as a protein translocation system Adaptation of Clostridium difficile toxin A for use as a protein translocation system](/preview/png/1931160.png)
A cellular delivery system is a useful biotechnology tool, with many possible applications. Two derivatives of Clostridium difficile toxin A (TcdA) have been constructed (GFP-TcdA and Luc-TcdA), by fusing reporter genes to functional domains of TcdA, and evaluated for their ability to translocate their cargo into mammalian cells. The cysteine protease and receptor binding domains of TcdA have been examined and found to be functional when expressed in the chimeric construct. Whereas GFP failed to internalize in the context of the TcdA fusion, significant cellular luciferase activity was detected in vero cell lysates after treatment with Luc-TcdA. Treatment with bafilomycin A1, which inhibits endosomal acidification, traps the luciferase activity within endosomes. To further understand these results, clarified lysates were subjected to molecular weight sieving, demonstrating that active luciferase was released from Luc-TcdA after translocation and internal processing.
Research highlights
► Catalytic domain of TcdA was replaced by a luciferase reporter.
► Each functional domain retains activity in the context of the fusion protein.
► We provide evidence that reporter proteins are delivered into vero cells.
► System releases cargo into the cytosol, providing a powerful new biotechnology tool.
Journal: Biochemical and Biophysical Research Communications - Volume 405, Issue 4, 25 February 2011, Pages 570–574