Mammalian G-protein-coupled receptor expression in Escherichia coli: I. High-throughput large-scale production as inclusion bodies.
G-protein-coupled receptors (GPCRs) represent approximately 3% of human proteome and the most prominent class of pharmacological targets. Despite their important role in many functions, only the X-ray structures of rhodopsin, and more recently of the beta(1)- and beta(2)-adrenergic receptors, have been resolved. Structural studies of GPCRs require that several tedious preliminary steps be fulfilled before setting up the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. Here we report on screening expression conditions of approximately 100 GPCRs in Escherichia coli with a view to obtain large amounts of inclusion bodies, a prerequisite to the subsequent refolding step. A set of optimal conditions, including appropriate vectors (Gateway pDEST17oi), strain (C43), and fermentation at high optical density, define the best first instance choice. Beyond this minimal setting, however, the rate of success increases significantly with the number of conditions tested. In contrast with experiments based on a single GPCR expression, our approach provides statistically significant results and indicates that up to 40% of GPCRs can be expressed as inclusion bodies in quantities sufficient for subsequent refolding, solubilization, and purification.
Michalke
Kerstin
K
Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS, and Universités of Marseille, Case 932, 163 Ave de Luminy, F-13288 Marseille Cedex 09, France.
Gravière
Marie-Eve
ME
Huyghe
Céline
C
Vincentelli
Renaud
R
Wagner
Renaud
R
Pattus
Franc
F
Schroeder
Kathrin
K
Oschmann
Jan
J
Rudolph
Rainer
R
Cambillau
Christian
C
Desmyter
Aline
A
D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D008322:Mammals; D015202:Protein Engineering / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D043562:Receptors, G-Protein-Coupled