title: Cell-free production of G protein-coupled receptors for functional and structural studies.
abstract: G-protein coupled receptors (GPCRs) are key elements in signal transduction pathways of eukaryotic cells and they play central roles in many human diseases. So far, most structural and functional approaches have been limited by the immense difficulties in the production of sufficient amounts of protein samples in conventional expression systems based on living cells. We report the high level production of six different GPCRs in an individual cell-free expression system based on Escherichia coli extracts. The open nature of cell-free systems allows the addition of detergents in order to provide an artificial hydrophobic environment for the reaction. This strategy defines a completely new technique for the production of membrane proteins that can directly associate with detergent micelles upon translation. We demonstrate the efficient overproduction of the human melatonin 1B receptor, the human endothelin B receptor, the human and porcine vasopressin type 2 receptors, the human neuropeptide Y4 receptor and the rat corticotropin releasing factor receptor by cell-free expression. In all cases, the long chain polyoxyethylene detergent Brij78 was found to be highly effective for solubilization and milligram amounts of soluble protein could be generated in less than 24 h. Single particle analysis indicated a homogenous distribution of predominantly protein dimers of the cell-free expressed GPCR samples, with dimensions similar to the related rhodopsin. Ligand interaction studies with the endothelin B receptor and a derivative of its peptide ligand ET-1 gave further evidence of a functional folding of the cell-free produced protein.
authors.affiliation: Centre for Biomolecular Magnetic Resonance, University of Frankfurt/Main, Institute for Biophysical Chemistry, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Klammt
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwarz
authors.forename: Nora
authors.initials: N
authors.lastname: Eifler
authors.forename: Andreas
authors.initials: A
authors.lastname: Engel
authors.forename: Jacob
authors.initials: J
authors.lastname: Piehler
authors.forename: Winfried
authors.initials: W
authors.lastname: Haase
authors.forename: Steffen
authors.initials: S
authors.lastname: Hahn
authors.forename: Volker
authors.initials: V
authors.lastname: Dötsch
authors.forename: Frank
authors.initials: F
authors.lastname: Bernhard
mesh_terms: D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008081:Liposomes / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000648:ultrastructure; D012995:Solubility
chemical_list: D002457:Cell Extracts; D003902:Detergents; D008024:Ligands; D008081:Liposomes; D043562:Receptors, G-Protein-Coupled; D011092:Polyethylene Glycols; C043444:octadecyl polyoxyethylene ether