Selection and characterization of DARPins specific for the neurotensin receptor 1

Peter Milovnik, Davide Ferrari, Casim A. Sarkar, Andreas Plückthun

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)357-366
Number of pages10
JournalProtein Engineering, Design and Selection
Volume22
Issue number6
DOIs
StatePublished - Jun 2009
Externally publishedYes

Bibliographical note

Funding Information:
This work was supported by the NCCR Structural Biology. C.A.S. was supported by a postdoctoral fellowship from the National Institutes of Health (F32 GM069267). D.F was supported by an EMBO long-term fellowship (ALTF-129-2005).

Keywords

  • DARPins
  • GPCR
  • NTR1
  • Neurotensin
  • Ribosome display

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