title: Polymer-based cell-free expression of ligand-binding family B G-protein coupled receptors without detergents.
abstract: G-protein coupled receptors (GPCRs) constitute the largest family of intercellular signaling molecules and are estimated to be the target of more than 50% of all modern drugs. As with most integral membrane proteins (IMPs), a major bottleneck in the structural and biochemical analysis of GPCRs is their expression by conventional expression systems. Cell-free (CF) expression provides a relatively new and powerful tool for obtaining preparative amounts of IMPs. However, in the case of GPCRs, insufficient homogeneity of the targeted protein is a problem as the in vitro expression is mainly done with detergents, in which aggregation and solubilization difficulties, as well as problems with proper folding of hydrophilic domains, are common. Here, we report that using CF expression with the help of a fructose-based polymer, NV10 polymer (NVoy), we obtained preparative amounts of homogeneous GPCRs from the three GPCR families. We demonstrate that two GPCR B family members, corticotrophin-releasing factor receptors 1 and 2β are not only solubilized in NVoy but also have functional ligand-binding characteristics with different agonists and antagonists in a detergent-free environment as well. Our findings open new possibilities for functional and structural studies of GPCRs and IMPs in general.
authors.affiliation: Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Klammt
authors.forename: Marilyn H
authors.initials: MH
authors.lastname: Perrin
authors.forename: Innokentiy
authors.initials: I
authors.lastname: Maslennikov
authors.forename: Ludovic
authors.initials: L
authors.lastname: Renault
authors.forename: Martin
authors.initials: M
authors.lastname: Krupa
authors.forename: Witek
authors.initials: W
authors.lastname: Kwiatkowski
authors.forename: Henning
authors.initials: H
authors.lastname: Stahlberg
authors.forename: Wylie
authors.initials: W
authors.lastname: Vale
authors.forename: Senyon
authors.initials: S
authors.lastname: Choe
mesh_terms: D002474:Cell-Free System / Q000378:metabolism*; D003902:Detergents; D005630:Fructans / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular; D011108:Polymers / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D043562:Receptors, G-Protein-Coupled / Q000235:genetics* / Q000378:metabolism
chemical_list: D003902:Detergents; D005630:Fructans; D008024:Ligands; D011108:Polymers; D043562:Receptors, G-Protein-Coupled; C072599:levan