G-protein coupled receptor protein synthesis on a lipid bilayer using a reconstituted cell-free protein synthesis system

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

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish
Article number54
JournalLife
Volume8
Issue number4
DOIs
Publication statusPublished - 2018 Dec 1
Externally publishedYes

Fingerprint

protein synthesis
lipid bilayers
Lipid bilayers
Lipid Bilayers
G-Protein-Coupled Receptors
membrane proteins
lipids
Membrane Proteins
lipid
proteins
protein
cells
membrane
membranes
Proteins
Cell-Free System
Micelles
micelles
circular dichroism spectroscopy
Lipids

Keywords

  • Artificial cell
  • Cell-free protein synthesis
  • G-protein coupled receptor
  • Lipid bilayer
  • Lipid nanodisc
  • Membrane protein

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Biochemistry, Genetics and Molecular Biology(all)
  • Space and Planetary Science
  • Palaeontology

Cite this

G-protein coupled receptor protein synthesis on a lipid bilayer using a reconstituted cell-free protein synthesis system. / Gessesse, Belay; Nagaike, Takashi; Nagata, Koji; Shimizu, Yoshihiro; Ueda, Takuya.

In: Life, Vol. 8, No. 4, 54, 01.12.2018.

Research output: Contribution to journalArticle

Gessesse, Belay ; Nagaike, Takashi ; Nagata, Koji ; Shimizu, Yoshihiro ; Ueda, Takuya. / G-protein coupled receptor protein synthesis on a lipid bilayer using a reconstituted cell-free protein synthesis system. In: Life. 2018 ; Vol. 8, No. 4.
@article{f3bfc30e0b3a4f039d18e16a22f8a60a,
title = "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 = "Artificial cell, Cell-free protein synthesis, G-protein coupled receptor, Lipid bilayer, Lipid nanodisc, Membrane protein",
author = "Belay Gessesse and Takashi Nagaike and Koji Nagata and Yoshihiro Shimizu and Takuya Ueda",
year = "2018",
month = "12",
day = "1",
doi = "10.3390/life8040054",
language = "English",
volume = "8",
journal = "Life",
issn = "0024-3019",
publisher = "Multidisciplinary Digital Publishing Institute (MDPI)",
number = "4",

}

TY - JOUR

T1 - G-protein coupled receptor protein synthesis on a lipid bilayer using a reconstituted cell-free protein synthesis system

AU - Gessesse, Belay

AU - Nagaike, Takashi

AU - Nagata, Koji

AU - Shimizu, Yoshihiro

AU - Ueda, Takuya

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

AB - 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.

KW - Artificial cell

KW - Cell-free protein synthesis

KW - G-protein coupled receptor

KW - Lipid bilayer

KW - Lipid nanodisc

KW - Membrane protein

UR - http://www.scopus.com/inward/record.url?scp=85057231342&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85057231342&partnerID=8YFLogxK

U2 - 10.3390/life8040054

DO - 10.3390/life8040054

M3 - Article

AN - SCOPUS:85057231342

VL - 8

JO - Life

JF - Life

SN - 0024-3019

IS - 4

M1 - 54

ER -