Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies
Keyword(s):
E Coli
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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.
2006 ◽
Vol 905
(1)
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pp. 25-33
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2002 ◽
Vol 1582
(1-3)
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pp. 72-80
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Keyword(s):
1998 ◽
Vol 13
(5)
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pp. 231-240
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2002 ◽
Vol 136
(1)
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pp. 9-22
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1994 ◽
Vol 3
(4)
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pp. 371-378
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