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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Escherichia coli / metabolism*
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Escherichia coli Proteins / metabolism*
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Inclusion Bodies / metabolism*
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Protein Refolding*
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Receptors, G-Protein-Coupled / metabolism*
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Sphingosine-1-Phosphate Receptors / metabolism*
Substances
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Escherichia coli Proteins
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Receptors, G-Protein-Coupled
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Sphingosine-1-Phosphate Receptors
Grants and funding
This study was supported by the Cooperative Research Training Group Pharmaceutical Biotechnology stated by the Postgraduate Scholarships Act of the Ministry for Science Research and Arts of the federal state government of Baden-Württemberg, Germany. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.