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

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.

The ligand binding ability of dopamine D1 receptors synthesized using a wheat germ cell-free protein synthesis system with liposomes

“…Assuming the molecular weight of human dopamine D 1 receptor estimated from amino acid numbers as 49,300, the product of dopamine D 1 receptor was calculated to be 6.49 nmol/mg of total protein, whereas an extremely low B max value was obtained. There are several papers indicating that wild-type GPCRs expressed in a cell-free protein synthesis system lack ligand binding activity (Yang et al 2011). Our experiments suggest that only a small fraction, that is, 0.02% of the total number of receptors, might participate in ligand binding.…”

“…To circumvent these problems, trials have been performed to insert T4 bacteriophage lysozyme sequences into the third intracellular loop in order to stabilize the receptor, which recovered the binding activities of β 2 -adrenoceptor (Yang et al, 2011) and histamine H 1 receptor (Shimamura et al, 2011). Yang et al (2011) also pointed out that oxidative protein folding is required for ligand binding. In human β 2 -adrenoceptor the extracellular disulfide bond between cysteines at position 184 and 190 is important to maintain binding activity.…”

Advances in the use of nanoscale bilayers to study membrane protein structure and function

“…Several groups have successfully used CF systems to incorporate MPs into nanodiscs that were pre-assembled and added to the translation reaction (Katzen et al, 2008;Lyukmanova et al, 2012;Yang, Cirico, Katzen, Peterson, & Kudlicki, 2011) or to co-express the target protein and MSP in the presence of phospholipids, thereby enabling the in situ assembly of the MP-containing nanodisc . The technical advantages of using such CF expression systems are manifold: (i) the potential cytotoxicity and subsequent low yields associated with in vivo expression of some MPs is avoided; (ii) there is no requisite detergent solubilization of the target MP; (iii) CF expression systems are open, facilitating the addition of cofactors and site-specific labeling of the MP with reporters such as fluorescent and EPR probes for upstream analyses.…”

Cell-free expression of a mammalian olfactory receptor and unidirectional insertion into small unilamellar vesicles (SUVs)

“…N-linked glycosylation of OR5 on the conserved NXS/T sequence at the N-terminus, and different insertion mechanisms (like co-or posttranslational insertion), which we did not further address in this study. Today, most membrane proteins and specially GPCRs are expressed by bacteria related cell-free expression systems [18,24] optimized for high protein yields. However, based on the highest yield of OR5 membrane insertion and concerning the genetic origin of mammalian OR5, the RRL system was selected for further studies.…”

“…Alternatively, reconstitution of membrane proteins into lipid membranes results in a random distribution and reduced accessibility to ligands [14,15]. Currently, cell-free approaches showed the expression and co-translational insertion of GPCRs directly into a lipid bilayer [16,17], in "nanodiscs" which are discoidal phospholipid bilayers stabilized by amphiphilic helical membrane proteins [18] or even in amphiphilic block-copolymer membranes [19]. In terms of the cotranslational insertion into lipid bilayers or liposomes, a unidirectional orientation of the GPCR molecules in the membrane structure is observed, since the protein synthesis machinery is added to the exposed plane of the bilayer membrane surface [20].…”