improved in vitro folding of the y2 g protein-coupled receptor into bicelles

Clicks: 210
ID: 249478
2018
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Abstract
Prerequisite for structural studies on G protein-coupled receptors is the preparation of highly concentrated, stable, and biologically active receptor samples in milligram amounts of protein. Here, we present an improved protocol for Escherichia coli expression, functional refolding, and reconstitution into bicelles of the human neuropeptide Y receptor type 2 (Y2R) for solution and solid-state NMR experiments. The isotopically labeled receptor is expressed in inclusion bodies and purified using SDS. We studied the details of an improved preparation protocol including the in vitro folding of the receptor, e.g., the native disulfide bridge formation, the exchange of the denaturating detergent SDS, and the functional reconstitution into bicelle environments of varying size. Full pharmacological functionality of the Y2R preparation was shown by a ligand affinity of 4 nM and G-protein activation. Further, simple NMR experiments are used to test sample quality in high micromolar concentration.
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schmidt2018frontiersimproved Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors ;Peter Schmidt;Brian J. Bender;Brian J. Bender;Anette Kaiser;Khushboo Gulati;Holger A. Scheidt;Heidi E. Hamm;Jens Meiler;Jens Meiler;Annette G. Beck-Sickinger;Daniel Huster
Journal biopsychosocial medicine
Year 2018
DOI
10.3389/fmolb.2017.00100
URL
http://journal.frontiersin.org/article/10.3389/fmolb.2017.00100/full
Keywords
gpcr folding nmrbiology (general)

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