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Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.


ABSTRACT: Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.

SUBMITTER: Heim B 

PROVIDER: S-EPMC7904181 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.

Heim Bastian B   Handrick René R   Hartmann Marcus D MD   Kiefer Hans H  

PloS one 20210224 2


Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refo  ...[more]

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