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

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: G-protein coupled receptor; artificial cell; cell-free protein synthesis; lipid bilayer; lipid nanodisc; membrane protein.

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 and non-purified CX₃CL1 to determine the binding affinity by SPR. The localization of CX₃CR1-sfGFP was probed by confocal microscopy.

Figure 2

Productivity and solubility of cell-free expressed GPCRs. The GPCRs were expressed in the PURE system containing [³⁵S]-Methionine in the presence or absence of nanodisc (ND+ or ND-, respectively). The productivity and solubility were subsequently quantified for CX₃CR1 (A), CCR5 (B) and CCR5-Rb (C). The soluble fraction was quantified by dividing the supernatant (S) by the total (T) amount of synthesized protein.

Figure 3

CD measurement of cell-free synthesized GPCRs. Secondary structures of PURE-synthesized CX₃CR1 (A) and CCR5-Rb (B) in the presence of a micelle (25 mg/mL brain polar lipid and 75 mg/mL digitonin) were analyzed. For CD measurement, the detergent was exchanged with a buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% DDM and 0.002% CHS during Ni²⁺ column chromatography.

Figure 4

Receptor (CX₃CR1)-ligand (CX₃CL1) interaction in different environments. Schematic representation of receptor-ligand interaction in nanodisc (A) and micelle (B). In both (A,B), the upper, middle and lower figures correspond to the binding affinity constant, fitted kinetics and schematic representations of the immobilization strategy respectively. In all cases, the analytes at 2.5 nM, 5 nM, 10 nM, 40 nM and 80 nM concentrations were injected to determine the binding affinity constant using single cycle kinetics. For the nanodisc system, CX₃CL1 protein is denoted as the analyte and for the micelle system, His-tagged CX₃CR1 is denoted as the analyte.

Figure 5

Membrane localization of cell-free synthesized receptor protein. (A) Spontaneous membrane integration of CX₃CR1-sfGFP synthesized inside GUV. Four different representative GUV images taken under a different field of vision were presented. (B) sfGFP synthesized inside GUV as a control. Unlike CX₃CR1-sfGFP, sfGFP was localized in the lumen of the GUVs. The scale bars are 10 µm.

Figure 6

Analysis of CX₃CR1-nanodisc complex by gel filtration and negative staining. (A) Elution pattern of the CX₃CR1-nanodisc complex (red) and empty nanodisc (black) as observed during the size exclusion chromatography. The peak was normalized to the elution profile of empty nanodisc. (B) SDS-PAGE analysis of the elution fraction stained by Coomassie Brilliant Blue (CBB). (C) Homogeneity of CX₃CR1-nanodisc complex analyzed by transmission electron. The scale bar is 50 nm.