Simon, Manuel; Zangemeister-Wittke, Uwe; Plückthun, Andreas (2012). Facile double-functionalization of designed ankyrin repeat proteins using click and thiol chemistries. Bioconjugate chemistry, 23(2), pp. 279-86. Washington, D.C.: American Chemical Society 10.1021/bc200591x
Full text not available from this repository. (Request a copy)Click chemistry is a powerful technology for the functionalization of therapeutic proteins with effector moieties, because of its potential for bio-orthogonal, regio-selective, and high-yielding conjugation under mild conditions. Designed Ankyrin Repeat Proteins (DARPins), a novel class of highly stable binding proteins, are particularly well suited for the introduction of clickable methionine surrogates such as azidohomoalanine (Aha) or homopropargylglycine (Hpg), since the DARPin scaffold can be made methionine-free by an M34L mutation in the N-cap which fully maintains the biophysical properties of the protein. A single N-terminal azidohomoalanine, replacing the initiator Met, is incorporated in high yield, and allows preparation of "clickable" DARPins at about 30 mg per liter E. coli culture, fully retaining stability, specificity, and affinity. For a second modification, we introduced a cysteine at the C-terminus. Such DARPins could be conveniently site-specifically linked to two moieties, polyethylene glycol (PEG) to the N-terminus and the fluorophore Alexa488 to the C-terminus. We present a DARPin selected against the epithelial cell adhesion molecule (EpCAM) with excellent properties for tumor targeting as an example. We used these doubly modified molecules to measure binding kinetics on tumor cells and found that PEGylation has no effect on dissociation rate, but slightly decreases the association rate and the maximal number of cell-bound DARPins, fully consistent with our previous model of PEG action obtained in vitro. Our data demonstrate the benefit of click chemistry for site-specific modification of binding proteins like DARPins to conveniently add several functional moieties simultaneously for various biomedical applications.
Item Type: |
Journal Article (Original Article) |
|---|---|
Division/Institute: |
04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Pharmacology |
UniBE Contributor: |
Simon, Manuel, Zangemeister-Wittke, Uwe |
Subjects: |
600 Technology > 610 Medicine & health |
ISSN: |
1043-1802 |
Publisher: |
American Chemical Society |
Language: |
English |
Submitter: |
Factscience Import |
Date Deposited: |
04 Oct 2013 14:35 |
Last Modified: |
05 Dec 2022 14:11 |
Publisher DOI: |
10.1021/bc200591x |
PubMed ID: |
22188139 |
Web of Science ID: |
000300213400014 |
URI: |
https://boris.unibe.ch/id/eprint/14100 (FactScience: 220908) |
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