Home Life Sciences Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration
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Oxidative in vitro folding of a cysteine deficient variant of the G protein-coupled neuropeptide Y receptor type 2 improves stability at high concentration

  • Kristina Witte , Anette Kaiser , Peter Schmidt EMAIL logo , Victoria Splith , Lars Thomas , Sandra Berndt , Daniel Huster and Annette G. Beck-Sickinger
Published/Copyright: June 4, 2013

Abstract

In vitro folding of G protein-coupled receptors into a detergent environment represents a promising strategy for obtaining sufficient amounts of functional receptor molecules for structural studies. Typically, these preparations exhibit a poor long-term stability especially at the required high protein concentration. Here, we report a protocol for the stabilization of the Escherichia coli-expressed and subsequently folded neuropeptide Y receptor type 2. We identified the free cysteines in the receptor as one major reason for intermolecular protein aggregation. Therefore, six out of the eight cysteine residues were mutated to alanine or serine without any significant loss of functionality of the receptor as demonstrated in cell culture models. Furthermore, the disulfide bond between the remaining two cysteines was irreversibly formed by applying oxidative in vitro folding. Applying this strategy, the stability of the functionally folded Y2 receptor could be increased to 20 days at a concentration of 15 μm in a micelle environment consisting of 1,2-diheptanoyl-sn-glycero-3-phosphocholine and n-dodecyl-ß-D-maltoside.


Corresponding author: Peter Schmidt, Institute for Medical Physics and Biophysics, Medical Department, Universität Leipzig, Härtelstrasse 16-18, D-04107 Leipzig, Germany

The authors thank Stefanie Babilon for assistance with receptor solubilization and analysis from eukaryotic cells. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 610, Projects A1 and A14) and by the ‘Nachwuchsförderung’ of the Medical Department, University of Leipzig.

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Received: 2013-1-15
Accepted: 2013-5-31
Published Online: 2013-06-04
Published in Print: 2013-08-01

©2013 by Walter de Gruyter Berlin Boston

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