Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies.
| Autor: | Heim B; Institute of Applied Biotechnology, University of Applied Sciences, Biberach, Germany., Handrick R; Institute of Applied Biotechnology, University of Applied Sciences, Biberach, Germany., Hartmann MD; Max Planck Institute for Developmental Biology, Tübingen, Germany., Kiefer H; Institute of Applied Biotechnology, University of Applied Sciences, Biberach, Germany. |
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| Jazyk: | angličtina |
| Zdroj: | PloS one [PLoS One] 2021 Feb 24; Vol. 16 (2), pp. e0247689. Date of Electronic Publication: 2021 Feb 24 (Print Publication: 2021). |
| DOI: | 10.1371/journal.pone.0247689 |
| Abstrakt: | 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. Competing Interests: The authors have declared that no competing interests exist. |
| Databáze: | MEDLINE |
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