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A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli


Egloff, Pascal; Deluigi, Mattia; Heine, Philipp; Balada, Stefanie; Plückthun, Andreas (2015). A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli. Protein Expression and Purification, 108:106-114.

Abstract

G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

Abstract

G protein-coupled receptors (GPCRs) are key players of cell signaling, thus representing important drug targets for the treatment of human diseases. Since inherent difficulties in receptor production and handling have precluded the application of many in vitro experiments, major questions about GPCR mechanisms and dynamics remain elusive to date. We recently used directed evolution in Escherichia coli on neurotensin receptor 1 (NTR1) for the generation of GPCR variants with greatly elevated functional expression levels and with excellent stability in detergent micelles. In this work we outline a highly efficient purification method for our evolved receptor variants, which is based on the application of an inexpensive, disposable high-affinity ligand column as the initial purification step. The ligand resin allows isolation of correctly folded GPCR variants directly from whole E. coli cell lysates at the scale of 10mg and it permits preparations of agonist- and antagonist-bound receptor samples. The purification principle presented here was key to the first structures of signaling-active NTR1 variants (Egloff et al., 2014). Since E. coli is uniquely suitable for the production of fully deuterated proteins, our method provides the basis for an array of NMR experiments that were not feasible for GPCRs to date, but which will shed light on novel aspects of receptor function and dynamics.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Department of Biochemistry
07 Faculty of Science > Department of Biochemistry
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:April 2015
Deposited On:24 Sep 2015 12:52
Last Modified:05 Apr 2016 19:25
Publisher:Elsevier
ISSN:1046-5928
Publisher DOI:https://doi.org/10.1016/j.pep.2014.10.006
PubMed ID:25461958

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