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Journal Article

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

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Hartmann,  MD
Department Protein Evolution, Max Planck Institute for Developmental Biology, Max Planck Society;
Molecular Recognition and Catalysis Group, Department Protein Evolution, Max Planck Institute for Developmental Biology, Max Planck Society;

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Citation

Heim, B., Handrick, R., Hartmann, M., & Kiefer, H. (2021). Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies. PLoS One, 16(2): e0247689. doi:10.1371/journal.pone.0247689.


Cite as: https://hdl.handle.net/21.11116/0000-000A-51E7-6
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.