Selection and characterization of DARPins specific for the neurotensin receptor 1.
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 microg 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.
Milovnik
Peter
P
Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
Ferrari
Davide
D
Sarkar
Casim A
CA
Plückthun
Andreas
A
D000818:Animals; D017089:Ankyrin Repeat / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000235:genetics; D008856:Microscopy, Fluorescence; D019151:Peptide Library; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D011869:Radioligand Assay; D051381:Rats; D018028:Receptors, Neurotensin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D019151:Peptide Library; D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; C114036:neurotensin type 1 receptor