Open AccessRefolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies
Author(s) -
Bastian Heim,
René Handrick,
M.D. Hartmann,
H. Kiefer
Publication year - 2021
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0247689
Subject(s) - g protein coupled receptor , thermostability , receptor , inclusion bodies , chemistry , biochemistry , biophysics , escherichia coli , biology , gene , enzyme
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 S1P 1 , and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.