UZH-Logo

Maintenance Infos

Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems


Verdurmen, Wouter P R; Luginbühl, Manuel; Honegger, Annemarie; Plückthun, Andreas (2015). Efficient cell-specific uptake of binding proteins into the cytoplasm through engineered modular transport systems. Journal of Controlled Release, 200:13-22.

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.

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.

Citations

12 citations in Web of Science®
12 citations in Scopus®
Google Scholar™

Altmetrics

Downloads

0 downloads since deposited on 22 Jan 2015
0 downloads since 12 months

Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:28 February 2015
Deposited On:22 Jan 2015 13:14
Last Modified:05 Apr 2016 18:54
Publisher:Elsevier
ISSN:0168-3659
Publisher DOI:https://doi.org/10.1016/j.jconrel.2014.12.019
PubMed ID:25526701
Permanent URL: https://doi.org/10.5167/uzh-106243

Download

[img]
Content: Published Version
Filetype: PDF - Registered users only
Size: 1MB
View at publisher

TrendTerms

TrendTerms displays relevant terms of the abstract of this publication and related documents on a map. The terms and their relations were extracted from ZORA using word statistics. Their timelines are taken from ZORA as well. The bubble size of a term is proportional to the number of documents where the term occurs. Red, orange, yellow and green colors are used for terms that occur in the current document; red indicates high interlinkedness of a term with other terms, orange, yellow and green decreasing interlinkedness. Blue is used for terms that have a relation with the terms in this document, but occur in other documents.
You can navigate and zoom the map. Mouse-hovering a term displays its timeline, clicking it yields the associated documents.

Author Collaborations