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

Takuya Ueda,Takashi Nagaike,Yoshihiro Shimizu,Belay Gessesse,Koji Nagata

doi:10.3390/life8040054

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.

Highlights

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
To understand the functional and structural roles of a repertoire of G-protein coupled receptors (GPCRs), we systematically examined their expression and productivity by the PURE system containing lipid nanodiscs
We supplemented the PURE system with lipid bilayers such as nanodiscs and evaluated the expression of the G-protein coupled receptors CX3 CR1 and Chemokine receptor type 5 (CCR5)

Summary

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. 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]. 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 be extended to other classes of membrane proteins such as GPCRs which can respond to extracellular signals in the environment. The synthesis and integration of active GPCRs into artificial lipid bilayer

Methods

Results

Conclusion