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G-Protein Coupled Receptor Protein Synthesis on a Lipid Bilayer Using a Reconstituted Cell-Free Protein Synthesis System

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G-Protein Coupled Receptor Protein Synthesis on a Lipid Bilayer Using a Reconstituted Cell-Free Protein Synthesis System life Article G-Protein Coupled Receptor Protein Synthesis on a Lipid Bilayer Using a Reconstituted Cell-Free Protein Synthesis System Belay Gessesse 1,2, Takashi Nagaike 1, Koji Nagata 3, Yoshihiro Shimizu 2 and Takuya Ueda 1,* 1 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bldg. FSB-401, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan; [email protected] (B.G.); [email protected] (T.N.) 2 Laboratory for Cell-Free Protein Synthesis, RIKEN Center for Biosystems Dynamics Research (BDR), 6-2-3, Furuedai, Suita, Osaka 565-0874, Japan; [email protected] 3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan; [email protected] * Correspondence: [email protected] Received: 31 August 2018; Accepted: 30 October 2018; Published: 2 November 2018 Abstract: Membrane proteins are important drug targets which play a pivotal role in various cellular activities. However, unlike cytosolic proteins, most of them are difficult-to-express proteins. In this study, to synthesize and produce sufficient quantities of membrane proteins for functional and structural analysis, we used a bottom-up approach in a reconstituted cell-free synthesis system, the PURE system, supplemented with artificial lipid mimetics or micelles. Membrane proteins were synthesized by the cell-free system and integrated into lipid bilayers co-translationally. Membrane proteins such as the G-protein coupled receptors were expressed in the PURE system and a productivity ranging from 0.04 to 0.1 mg per mL of reaction was achieved with a correct secondary structure as predicted by circular dichroism spectrum. In addition, a ligand binding constant of 27.8 nM in lipid nanodisc and 39.4 nM in micelle was obtained by surface plasmon resonance and the membrane protein localization was confirmed by confocal microscopy in giant unilamellar vesicles. We found that our method is a promising approach to study the different classes of membrane proteins in their native-like artificial lipid bilayer environment for functional and structural studies. Keywords: cell-free protein synthesis; artificial cell; lipid bilayer; membrane protein; G-protein coupled receptor; lipid nanodisc 1. Introduction The advent of artificial cells has enabled us to study active membrane proteins that play a substantial role in various cellular activities and physiological functions under a defined condition [1,2]. Membrane proteins are expressed and localized to the biological membrane to modulate the functions of the membrane by regulating the bidirectional flux of molecules and cell-to-cell communications. It is estimated that about 30–50% of all pharmaceutical drugs target membrane proteins such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters and enzymes [3–6]. The synthesis of active membrane proteins using artificial lipid bilayers has a significant advantage in examining the complex biochemical reactions and to screen for drug candidates. Artificial cells have been manipulated to study the functions of membrane proteins such as α-hemolysin [7,8], a potassium channel, KcsA [9] and olfactory receptor complexes [10]. The use of artificial cells can also be extended to other classes of membrane proteins such as GPCRs which can respond to extracellular signals in the environment. Therefore, the synthesis and integration of active GPCRs into artificial lipid bilayer Life 2018, 8, 54; doi:10.3390/life8040054 www.mdpi.com/journal/life Life 2018, 8, 54 2 of 14 mimetics such as nanodiscs and giant unilamellar vesicles (GUVs) have a substantial role to screen ligands and to develop biosensors for medical applications [11]. Currently, cell-free protein synthesis systems have become an emerging potential bottom-up approach to study membrane proteins for functional and structural analysis. The Protein Synthesis Using Recombinant Elements system (PURE system) is an E. coli based cell-free system which contains purified components of transcriptional and translational factors at a defined concentration unlike the total E. coli extractLife 2018 based, 8, x FOR cell-free PEER REVIEW system [12]. This enables us to control and modify2 of 14 the system more easily as per ourartificial objectives. cells can also Moreover, be extended to the other PURE classes systemof membrane is proteins devoid such of as nucleases GPCRs which and can proteases and offers improvedrespond controllability. to extracellular signals To date, in the various environment. functional Therefore, the membrane synthesis and proteinsintegration of were active synthesized by GPCRs into artificial lipid bilayer mimetics such as nanodiscs and giant unilamellar vesicles (GUVs) the cell-free systemshave a substantial supplemented role to screen with ligands detergents, and to develop chaperones, biosensors for medical micelles, applications liposomes [11]. and nanodiscs. These include C-CCurrently, Chemokine cell-free receptorprotein synt typehesis systems 5 (CCR5) have become [13], claudin-4an emerging [potential14], secYEG bottom-up [15 ] and human approach to study membrane proteins for functional and structural analysis. The Protein Synthesis endothelin B receptorUsing Recombinant (ETB) [16 Elements]. system (PURE system) is an E. coli based cell-free system which Incorporationcontains of purifiedlipid components nanodiscs of transcriptional (NDs) into and thetranslational cell-free factors system at a defined during concentration membrane protein expression has aunlike greater the total advantage E. coli extract over based vesicle-based cell-free system [12]. mimetics This enables such us to as control GUVs. and Nanodiscsmodify the are generally system more easily as per our objectives. Moreover, the PURE system is devoid of nucleases and soluble, stable, monodisperseproteases and offers and improved more cont importantly,rollability. To date, both various the N- func andtional C-terminus membrane proteins are accessible were for ligand binding assays [synthesized17,18]. Recently, by the cell-free the systems nanodisc supplemented technology with detergents, has been chaperones, used for micelles, the reconstitution liposomes of various and nanodiscs. These include C-C Chemokine receptor type 5 (CCR5) [13], claudin-4 [14], secYEG membrane proteins[15] and such human as endothelin a G-protein B receptor coupled (ETB) [16]. receptor, CCR5 [19], an ion channel TRPV1 [20] and a transporter, MsbAIncorporation [21]. In additionof lipid nanodiscs to the (NDs) direct into reconstitution the cell-free system of during purified membrane membrane protein proteins into expression has a greater advantage over vesicle-based mimetics such as GUVs. Nanodiscs are nanodiscs, co-translationalgenerally soluble, integration stable, monodisperse of membrane and more proteins importantly, by both a cell-free the N- and system C-terminus was are nonetheless used for adrenergic receptoraccessible for 1 ligand (β1AR) binding [22 assays] and [17,18]. endothelial Recently, the receptor nanodisc technology (ETB) [16 has,23 been]. used From for athe structural point of view, nanodiscsreconstitution in combination of various membrane with cell-free proteins such systems as a G-protein will offercoupled a receptor, greater CCR5 advantage [19], an ion in determining channel TRPV1 [20] and a transporter, MsbA [21]. In addition to the direct reconstitution of purified the structure ofmembrane membrane proteins proteinsinto nanodiscs, in co-translational their native-like integration lipid of membrane bilayer proteins by cryo-electron by a cell-free microscopy (cryo-EM) [21,24system]. was nonetheless used for adrenergic receptor 1 (β1AR) [22] and endothelial receptor (ETB) [16,23]. From a structural point of view, nanodiscs in combination with cell-free systems will offer a In this study,greater we advantageused a PUREin determining system the basedstructure co-translational of membrane proteins integration in their native-like of GPCRs lipid bilayer into artificial lipid bilayers and micellesby cryo-electron for functional microscopy and (cryo-EM) structural [21,24]. analysis. To gain an insight into the functions of GPCRs, In this study, we used a PURE system based co-translational integration of GPCRs into artificial lipid we chose the chemokinebilayers and micelles GPCRs, for functional CX3CR1 and and structural CCR5 anal asysis. model To gain an proteins. insight into Wethe functions systematically of GPCRs, optimized the expression andwe analyzed chose the chemokine the secondary GPCRs, CX structure,3CR1 and CCR5 membrane as model proteins. localization, We systematically activity optimized and the homogeneity of the synthesizedexpression chemokine and analyzed GPCRs the secondary by using structure, nanodiscs, membrane GUVs localization, and activity micelles and homogeneity (Figure1 ofA–C). the synthesized chemokine GPCRs by using nanodiscs, GUVs and micelles (Figure 1A–C). Figure 1. Schematic representation of the cell-free expression of GPCRs. CX3CR1 was synthesized by the PURE system supplemented with lipid nanodiscs (A), micelle (B), or CX3CR1-sfGFP synthesized inside GUV (C). The GPCR-nanodisc complex was immobilized on a sensor chip without prior purification for Surface Plasmon Resonance (SPR) based ligand binding assay. Similarly, PURE expressed CX3CR1 in micelle was purified and allowed to interact with pre-immobilized PURE expressed
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