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PEDS Advance Access published online on April 22, 2009
Protein Engineering Design and Selection, doi:10.1093/protein/gzp011
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Selection and characterization of DARPins specific for the neurotensin receptor 1

Peter Milovnik1,4, Davide Ferrari1,2,4, Casim A. Sarkar1,3 and Andreas Plückthun1,5

1 Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

5 To whom correspondence should be addressed. E-mail: plueckthun{at}bioc.uzh.ch

We describe here the selection and characterization of designed ankyrin repeat proteins (DARPins) that bind specifically to the rat neurotensin receptor 1 (NTR1), a G-protein coupled receptor (GPCR). The selection procedure using ribosome display and the initial clone analysis required <10 µg of detergent-solubilized, purified NTR1. Complex formation with solubilized GPCR was demonstrated by ELISA and size-exclusion chromatography; additionally, the GPCR could be detected in native membranes of mammalian cells using fluorescence microscopy. The main binding epitope in the GPCR lies within the 33 amino acids following the seventh transmembrane segment, which comprise the putative helix 8, and additional binding interactions are possibly contributed by the cytoplasmic loop 3, thus constituting a discontinuous epitope. Since the selected binders recognize the GPCR both in detergent-solubilized and in membrane-embedded forms, they will be potentially useful both in co-crystallization trials and for signal transduction experiments.

Keywords: DARPins/GPCR/neurotensin/NTR1/ribosome display

Received February 8, 2009; revised March 6, 2009; accepted March 15, 2009.

2 Present address: Dipartimento di Biochimica e Biologia Molecolare, Università di Parma, Via Usberti 23/a, 43100 Parma, Italy

3 Present address: Department of Bioengineering, University of Pennsylvania, 240 Skirkanich Hall, 210 S. 33rd St., Philadelphia, PA 19104-6321, USA

4 These authors contributed equally to this work.


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