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Toxins 2014, 6(5), 1644-1666; doi:10.3390/toxins6051644
Article

Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

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Received: 21 March 2014; in revised form: 6 May 2014 / Accepted: 8 May 2014 / Published: 22 May 2014
(This article belongs to the Special Issue Intracellular Traffic and Transport of Bacterial Protein Toxins)
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Abstract: Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM.
Keywords: endosomal escape; reporter assay; protein therapeutics; ribosome inactivating proteins; horseradish peroxidase; Alexa Fluor 488; ricin A-chain; saporin; triterpenoidal saponins endosomal escape; reporter assay; protein therapeutics; ribosome inactivating proteins; horseradish peroxidase; Alexa Fluor 488; ricin A-chain; saporin; triterpenoidal saponins
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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MDPI and ACS Style

Gilabert-Oriol, R.; Thakur, M.; von Mallinckrodt, B.; Bhargava, C.; Wiesner, B.; Eichhorst, J.; Melzig, M.F.; Fuchs, H.; Weng, A. Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics. Toxins 2014, 6, 1644-1666.

AMA Style

Gilabert-Oriol R, Thakur M, von Mallinckrodt B, Bhargava C, Wiesner B, Eichhorst J, Melzig MF, Fuchs H, Weng A. Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics. Toxins. 2014; 6(5):1644-1666.

Chicago/Turabian Style

Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F.; Fuchs, Hendrik; Weng, Alexander. 2014. "Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics." Toxins 6, no. 5: 1644-1666.


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