Affordable Access

Access to the full text

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

  • 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)
Published Article
BMC Biotechnology
Springer (Biomed Central Ltd.)
Publication Date
May 23, 2011
DOI: 10.1186/1472-6750-11-57
Springer Nature


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.

Report this publication


Seen <100 times