Selection and characterization of DARPins specific for the neurotensin receptor 1.
Selection and characterization of DARPins specific for the neurotensin receptor 1.
复制标题
神经降压素受体 1 特异性 DARPins 的选择和表征。
DOI：
10.1093/protein/gzp011
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Plückthun,Andreas
中科院分区：
文献类型：
--
作者：
Milovnik,Peter;Ferrari,Davide;Sarkar,CasimA;Plückthun,Andreas
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.
