title: Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli.
abstract: G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.
authors.affiliation: Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas 78712, USA.
authors.forename: A James
authors.initials: AJ
authors.lastname: Link
authors.forename: Georgios
authors.initials: G
authors.lastname: Skretas
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Strauch
authors.forename: Nandini S
authors.initials: NS
authors.lastname: Chari
authors.forename: George
authors.initials: G
authors.lastname: Georgiou
mesh_terms: D049069:ATP-Dependent Proteases / Q000096:biosynthesis / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000096:biosynthesis; D006801:Humans; D015202:Protein Engineering; D043782:Receptor, Bradykinin B2 / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D043884:Receptor, Cannabinoid, CB1 / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
chemical_list: D003902:Detergents; D029968:Escherichia coli Proteins; D043782:Receptor, Bradykinin B2; D043884:Receptor, Cannabinoid, CB1; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli