title: A cleavable ligand column for the rapid isolation of large quantities of homogeneous and functional neurotensin receptor 1 variants from E. coli.
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.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Pascal
authors.initials: P
authors.lastname: Egloff
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Mattia
authors.initials: M
authors.lastname: Deluigi
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Philipp
authors.initials: P
authors.lastname: Heine
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Balada
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland. Electronic address: plueckthun@bioc.uzh.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018028:Receptors, Neurotensin / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018028:Receptors, Neurotensin; D011994:Recombinant Proteins; C114036:neurotensin type 1 receptor
keywords: Crystal structure; Directed evolution; G protein-coupled receptor; Ligand affinity purification; Membrane protein; Protein stability