Abstract
The C2 toxin produced by Clostridium botulinum is a binary AB-type exotoxin composed of the enzyme subunit C2I and the binding/translocation moiety C2II. After proteolytic activation, C2IIa mediates the subsequent internalization of C2I into the cytosol of mammalian target cells. The N-terminal domain of C2I (C2IN) is necessary for C2IIa-dependent uptake, but lacks the enzyme domain that is responsible for cytotoxicity. In the present study, we generated a delivery system building on C2IN and a truncated core streptavidin (Stv13) with enhanced solubility for the C2IIa-dependent internalization of biotinylated cargo molecules into mammalian cells. C2IN–Stv13 fusion protein expressed in Escherichia coli was obtained in high yields and purity. The affinity-purified protein formed tetramers and a defined higher order species in solution as shown by gel filtration and retained its biotin-binding properties, however with an obvious reduction in affinity. Uptake of C2IN–Stv13 into the cytosol of HeLa and other cancer cell lines was observed by immunoblot analysis, which was corroborated by confocal microscopy. In addition, the fusion protein was not cytotoxic and did not inhibit cell proliferation as determined by MTS assay. Finally, we demonstrated the C2IN–Stv13/C2IIa-mediated uptake of biocytin–Alexa 488 as cargo into HeLa cells, underscoring the functionality of the generated transport system.
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Acknowledgments
We are grateful to Dr. Ulrich Pannicke, Institute for Transfusion Medicine, University of Ulm, Germany, for the kind gift of Jurkat T-cells, MOLT-3, and HepG2 cells. We would also like to thank Dr. Angelika Rück, ILM, University of Ulm, Germany, for providing access to the confocal microscope facility. We are indebted to Ulrike Binder for excellent technical assistance.
This work was funded by grant of the Medical Faculty, University of Ulm (Bausteinprojekt LSBN.0060 to J.F.)
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The authors declare that they have no conflict of interest.
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Fahrer, J., Funk, J., Lillich, M. et al. Internalization of biotinylated compounds into cancer cells is promoted by a molecular Trojan horse based upon core streptavidin and clostridial C2 toxin. Naunyn-Schmied Arch Pharmacol 383, 263–273 (2011). https://doi.org/10.1007/s00210-010-0585-7
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DOI: https://doi.org/10.1007/s00210-010-0585-7