title: A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.
abstract: Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.
authors: Center for Biomedical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors: Karolina
authors: K
authors: Corin
authors: Philipp
authors: P
authors: Baaske
authors: Deepali B
authors: DB
authors: Ravel
authors: Junyao
authors: J
authors: Song
authors: Emily
authors: E
authors: Brown
authors: Xiaoqiang
authors: X
authors: Wang
authors: Sandra
authors: S
authors: Geissler
authors: Christoph J
authors: CJ
authors: Wienken
authors: Moran
authors: M
authors: Jerabek-Willemsen
authors: Stefan
authors: S
authors: Duhr
authors: Dieter
authors: D
authors: Braun
authors: Shuguang
authors: S
authors: Zhang
mesh_terms: D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008024:Ligands; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D012995:Solubility
chemical_list: D008024:Ligands; D043562:Receptors, G-Protein-Coupled