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

BMC Biotechnol. 2011 May 23;11:57. doi: 10.1186/1472-6750-11-57.

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.

MeSH terms

  • Adrenergic beta-2 Receptor Antagonists / pharmacology
  • Bacteriophage T4 / enzymology
  • Cell-Free System
  • Cloning, Molecular
  • Dihydroalprenolol / pharmacology
  • Humans
  • Lipid Bilayers / chemistry
  • Lipid Bilayers / metabolism*
  • Muramidase / biosynthesis
  • Muramidase / genetics
  • Nanostructures / chemistry
  • Protein Binding
  • Protein Folding
  • Receptors, Adrenergic, beta-2 / biosynthesis*
  • Receptors, Adrenergic, beta-2 / chemistry
  • Receptors, Adrenergic, beta-2 / genetics
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / genetics

Substances

  • Adrenergic beta-2 Receptor Antagonists
  • Lipid Bilayers
  • Receptors, Adrenergic, beta-2
  • Recombinant Fusion Proteins
  • Dihydroalprenolol
  • Muramidase

Associated data

  • RefSeq/NM_000024
  • RefSeq/NM_000524
  • RefSeq/NM_000738