Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems.
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| Abstract |
Through advances in protein scaffold engineering and selection technologies, highly specific binding proteins, which fold under reducing conditions, can be generated against virtually all targets. Despite tremendous therapeutic opportunities, intracellular applications are hindered by difficulties associated with achieving cytosolic delivery, compounded by even correctly measuring it. Here, we addressed cytosolic delivery systematically through the development of a biotin ligase-based assay that objectively quantifies cytosolic delivery in a generic fashion. We developed modular transport systems that consist of a designed ankyrin repeat protein (DARPin) for receptor targeting and a different DARPin for intracellular recognition and a bacterial toxin-derived component for cytosolic translocation. We show that both anthrax pores and the translocation domain of Pseudomonas exotoxin A (ETA) efficiently deliver DARPins into the cytosol. We found that the cargo must not exceed a threshold thermodynamic stability for anthrax pores, which can be addressed by engineering, while the ETA pathway does not appear to have this restriction.
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| Authors | Wouter P R Verdurmen, Manuel Luginbühl, Annemarie Honegger, Andreas Plückthun |
| Journal | Journal of controlled release : official journal of the Controlled Release Society (J Control Release) Vol. 200 Pg. 13-22 (Feb 28 2015) ISSN: 1873-4995 [Electronic] Netherlands |
| PMID | 25526701 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't) |
| Copyright | Copyright © 2014 Elsevier B.V. All rights reserved. |
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