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Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA)
Authors:Christoph Beitzinger  Caroline Stefani  Angelika Kronhardt  Monica Rolando  Gilles Flatau  Emmanuel Lemichez  Roland Benz
Institution:1. Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.; 2. Toxines microbiennes dans la relation hôte-pathogènes, C3M, U1065, Inserm, Nice, France.; 3. UFR Médecine, IFR50, Université de Nice-Sophia Antipolis, Nice, France.; 4. School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.; Universidad Nacional Autonoma de Mexico, Instituto de Biotecnologia, Mexico,
Abstract:It is of interest to define bacterial toxin biochemical properties to use them as molecular-syringe devices in order to deliver enzymatic activities into host cells. Binary toxins of the AB7/8-type are among the most potent and specialized bacterial protein toxins. The B subunits oligomerize to form a pore that binds with high affinity host cell receptors and the enzymatic A subunit. This allows the endocytosis of the complex and subsequent injection of the A subunit into the cytosol of the host cells. Here we report that the addition of an N-terminal His6-tag to different proteins increased their binding affinity to the protective antigen (PA) PA63-channels, irrespective if they are related (C2I) or unrelated (gpJ, EDIN) to the AB7/8-family of toxins. His6-EDIN exhibited voltage-dependent increase of the stability constant for binding by a factor of about 25 when the trans-side corresponding to the cell interior was set to −70 mV. Surprisingly, the C. botulinum toxin C2II-channel did not share this feature of PA63. Cell-based experiments demonstrated that addition of an N-terminal His6-tag promoted also intoxication of endothelial cells by C2I or EDIN via PA63. Our results revealed that addition of His6-tags to several factors increase their binding properties to PA63 and enhance the property to intoxicate cells.
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