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Cell-free synthesis of a functional G protein-coupled receptor complexed with nanometer scale bilayer discs

Authors
  • Yang, Jian-Ping1
  • Cirico, Tatiana1
  • Katzen, Federico1
  • Peterson, Todd C1
  • Kudlicki, Wieslaw1
  • 1 Life Technologies, 5791 Van Allen Way, Carlsbad, CA, 92008, USA , Carlsbad (United States)
Type
Published Article
Journal
BMC Biotechnology
Publisher
Springer (Biomed Central Ltd.)
Publication Date
May 23, 2011
Volume
11
Issue
1
Identifiers
DOI: 10.1186/1472-6750-11-57
Source
Springer Nature
Keywords
License
Yellow

Abstract

BackgroundG 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.ResultsHere 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.ConclusionsAlthough 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.

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