Unknown

Dataset Information

0

Thermostability of a recombinant G protein-coupled receptor expressed at high level in mammalian cell culture.


ABSTRACT: Rational design of pharmaceutical drugs targeting integral membrane G protein-coupled receptors (GPCR) requires thorough understanding of ligand binding and mechanism of activation through high resolution structural studies of purified proteins. Due to inherent conformational flexibility of GPCR, stabilization of these proteins solubilized from cell membranes into detergents is a challenging task. Here, we take advantage of naturally occurring post-translational modifications for stabilization of purified GPCR in detergent micelles. The recombinant cannabinoid CB2 receptor was expressed at high yield in Expi293F mammalian cell cultures, solubilized and purified in Façade detergent. We report superior stability of the mammalian cell-expressed receptor compared to its E. coli-expressed counterpart, due to contributions from glycosylation of the N terminus and palmitoylation of the C terminus of CB2. Finally, we demonstrate that the mammalian Expi293F amino acid labelling kit is suitable for preparation of multi-milligram quantities of high quality, selectively stable isotope-labeled GPCR for studies by nuclear magnetic resonance.

SUBMITTER: Yeliseev A 

PROVIDER: S-EPMC7546613 | BioStudies | 2020-01-01

REPOSITORIES: biostudies

Similar Datasets

2009-01-01 | S-EPMC2689077 | BioStudies
2013-01-01 | S-EPMC3779079 | BioStudies
2017-01-01 | S-EPMC5406253 | BioStudies
1989-01-01 | S-EPMC1138903 | BioStudies
2012-01-01 | S-EPMC3463599 | BioStudies
2021-01-01 | S-EPMC7749421 | BioStudies
2019-01-01 | S-EPMC6688945 | BioStudies
2011-01-01 | S-EPMC3264755 | BioStudies
2008-01-01 | S-EPMC2531281 | BioStudies
1979-01-01 | S-EPMC1186636 | BioStudies