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PEDS Advance Access originally published online on March 24, 2005
Protein Engineering Design and Selection 2005 18(3):153-160; doi:10.1093/protein/gzi012
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oupjournals.org

Engineering and functional immobilization of opioid receptors

David Ott1, Yvonne Neldner, Régis Cèbe2, Igor Dodevski and Andreas Plückthun3

Biochemisches Institut, Universität Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. 1Present address: Universitätsspital Zürich, Departement Neuropathologie, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland. 2Present address: Novartis Pharma AG, WSJ 88/7.07, CH-4000 Basel, Switzerland

3 To whom correspondence should be addressed. E-mail: plueckthun{at}bioc.unizh.ch

Opioid receptors, like many G protein-coupled receptors (GPCRs), are notoriously unstable in detergents. We have now developed a more stable variant of the µ-opioid receptor (MOR) and also a method for the immobilization of solubilized, functional opioid receptors on a solid phase (magnetic beads). Starting with the intrinsically more stable {kappa}-opioid receptor (KOR), we optimized the conditions (i.e. detergents and stabilizing ligands) for receptor extraction from lipid bilayers of HEK293T cells to obtain maximal amounts of functional, immobilized receptor. After immobilization, the ligand binding profile remains the same as observed for the membrane-embedded receptor. For the immobilized wild-type µ-opioid receptor, however, no conditions were found under which ligand binding capacity was retained. To solve this problem, we engineered the receptor chimera KKM where the N-terminus and the first transmembrane helix (TM1) of wild-type MOR is exchanged for the homologous receptor parts of the wild-type KOR. This hybrid receptor behaves exactly as the wild-type MOR in functional assays. Interestingly, the modified MOR is expressed at six times higher levels than wild-type MOR and is similarly stable as wild-type KOR after immobilization. Hence the immobilized MOR, represented by the chimera KKM, is now also amenable for biophysical characterization. These results are encouraging for future stability engineering of GPCRs.

Keywords: functional immobilization/µ-opioid receptor/opioid receptors

Received February 15, 2005; accepted February 18, 2005.

Edited by David Eisenberg


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