title: Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs.
abstract: BACKGROUND
  G protein coupled receptors (GPCRs) represent the largest family of membrane proteins in the human genome and the richest source of targets for the pharmaceutical industry. A major limitation to characterizing GPCRs has been the difficulty in developing high-level heterologous expression systems that are cost effective. Reasons for these difficulties include inefficient transport and insertion in the plasma membrane and cytotoxicity. Additionally, GPCR purification requires detergents, which have a negative effect on receptor yields and stability.
  
  
  RESULTS
  Here we report a detergent-free cell-free protein expression-based method to obtain pharmacologically active GPCRs in about 2 hours. Our strategy relies on the co-translational insertion of modified GPCRs into nanometer-sized planar membranes. As a model we employed an engineered β2-adrenergic receptor in which the third intracellular loop has been replaced with T4 lysozyme (β2AR -T4L). We demonstrated that nanolipoprotein particles (NLPs) are necessary for expression of active β2AR -T4L in cell-free systems. The binding specificity of the NLP- β2AR-T4L complex has been determined by competitive assays. Our results demonstrate that β2AR-T4L synthesized in vitro depends on similar oxidative conditions as those required by an in vivo-expressed receptor.
  
  
  CONCLUSIONS
  Although the activation of β2AR-T4L requires the insertion of the T4 lysozyme sequence and the yield of that active protein limited, our results conceptually prove that cell-free protein expression could be used as a fast approach to express these valuable and notoriously difficult-to-express proteins.
authors: Life Technologies, 5791 Van Allen Way, Carlsbad, CA 92008, USA.
authors: Jian-Ping
authors: JP
authors: Yang
authors: Tatiana
authors: T
authors: Cirico
authors: Federico
authors: F
authors: Katzen
authors: Todd C
authors: TC
authors: Peterson
authors: Wieslaw
authors: W
authors: Kudlicki
references: Adv Exp Med Biol. 2009;611:333-5
references: 19400216
references: J Proteome Res. 2008 Feb;7(2):621-9
references: 18177001
references: J Biol Chem. 2002 Sep 13;277(37):34280-6
references: 12097322
references: Nature. 2007 Nov 15;450(7168):383-7
references: 17952055
references: FEBS J. 2007 Jul;274(13):3257-69
references: 17535295
references: Anal Biochem. 2005 Aug 15;343(2):344-6
references: 16005425
references: Science. 2007 Nov 23;318(5854):1266-73
references: 17962519
references: Structure. 2008 Jun;16(6):897-905
references: 18547522
references: Science. 2007 Nov 23;318(5854):1253-4
references: 18033872
references: J Biol Chem. 1994 Mar 4;269(9):6743-52
references: 8120034
references: Protein Expr Purif. 2005 May;41(1):27-37
references: 15802218
references: J Proteome Res. 2008 Aug;7(8):3535-42
references: 18557639
references: Science. 2008 Nov 21;322(5905):1211-7
references: 18832607
references: Science. 2007 Nov 23;318(5854):1258-65
references: 17962520
references: Mol Cell Proteomics. 2008 Nov;7(11):2246-53
references: 18603642
references: Nat Rev Microbiol. 2008 Mar;6(3):234-44
references: 18246081
references: Trends Biotechnol. 2009 Aug;27(8):455-60
references: 19616329
references: FEBS Lett. 2001 Aug 31;504(3):94-8
references: 11532439
references: Nature. 2008 Jul 24;454(7203):486-91
references: 18594507
references: J Struct Biol. 2007 Jun;158(3):482-93
references: 17350285
references: Mol Pharmacol. 2003 Jun;63(6):1202-5
references: 12761327
references: Biochemistry. 1990 Mar 6;29(9):2335-42
references: 2159799
references: Biotechnol Prog. 2007 May-Jun;23(3):540-7
references: 17397185
references: EMBO J. 2007 Apr 18;26(8):1995-2004
references: 17396152