Synthetic single domain antibodies for the conformational trapping of membrane proteins

Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, 
Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.
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Metadaten
Author:Iwan Zimmermann, Pascal Egloff, Cédric Hutter, Fabian M. Arnold, Peter Stohler, Nicolas Bocquet, Melanie N. Hug, Sylwia Huber, Martin Siegrist, Lisa Hetemann, Jennifer Gera, Samira Gmür, Peter Spies, Daniel Gygax, Eric R. Geertsma, Roger Dawson, Markus A. Seeger
URN:urn:nbn:de:hebis:30:3-503554
DOI:http://dx.doi.org/10.7554/eLife.34317.001
ISSN:2050-084X
Pubmed Id:http://www.ncbi.nlm.nih.gov/pubmed?term=29792401
Parent Title (English):eLife
Publisher:eLife Sciences Publications
Place of publication:Cambridge
Contributor(s):Lucy Forrest
Document Type:Article
Language:English
Year of Completion:2018
Date of first Publication:2018/05/24
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2019/06/13
Tag:Biochemistry and chemical biology; Conformational trapping; E. colo; In vitro selection; Membrane protein; Nanobody; Phage display; Ribosome display; Structural biology and molecular biophysics; Tools and ressources
Volume:7
Issue:e34317
Pagenumber:32
First Page:1
Last Page:32
Note:
Copyright Zimmermann et al. This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.
HeBIS PPN:450752313
Institutes:Biochemie und Chemie
Dewey Decimal Classification:570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0

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