Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses
Abstract Membrane proteins are key elements in cell-mediated processes. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. Up to date, there still exi...
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doaj-a5c74a40789144049b2f636de8b2e6d92020-12-08T00:18:00ZengNature Publishing GroupScientific Reports2045-23222017-06-017111010.1038/s41598-017-03955-8Qualifying a eukaryotic cell-free system for fluorescence based GPCR analysesAnne Zemella0Solveig Grossmann1Rita Sachse2Andrei Sonnabend3Michael Schaefer4Stefan Kubick5Fraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalysis and Bioprocesses, Potsdam-GolmRudolf-Boehm-Institut für Pharmakologie und Toxikologie, Medizinische FakultätFraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalysis and Bioprocesses, Potsdam-GolmFraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalysis and Bioprocesses, Potsdam-GolmRudolf-Boehm-Institut für Pharmakologie und Toxikologie, Medizinische FakultätFraunhofer Institute for Cell Therapy and Immunology (IZI), Branch Bioanalysis and Bioprocesses, Potsdam-GolmAbstract Membrane proteins are key elements in cell-mediated processes. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. Up to date, there still exist a number of GPCRs that has not been structurally and functionally analyzed due to difficulties in cell-based membrane protein production. A promising approach for membrane protein synthesis and analysis has emerged during the last years and is known as cell-free protein synthesis (CFPS). Here, we describe a simply portable method to synthesize GPCRs and analyze their ligand-binding properties without the requirement of additional supplements such as liposomes or nanodiscs. This method is based on eukaryotic cell lysates containing translocationally active endogenous endoplasmic reticulum-derived microsomes where the insertion of GPCRs into biologically active membranes is supported. In this study we present CFPS in combination with fast fluorescence-based screening methods to determine the localization, orientation and ligand-binding properties of the endothelin B (ET-B) receptor upon expression in an insect-based cell-free system. To determine the functionality of the cell-free synthesized ET-B receptor, we analyzed the binding of its ligand endothelin-1 (ET-1) in a qualitative fluorescence-based assay and in a quantitative radioligand binding assay.https://doi.org/10.1038/s41598-017-03955-8 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Anne Zemella Solveig Grossmann Rita Sachse Andrei Sonnabend Michael Schaefer Stefan Kubick |
spellingShingle |
Anne Zemella Solveig Grossmann Rita Sachse Andrei Sonnabend Michael Schaefer Stefan Kubick Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses Scientific Reports |
author_facet |
Anne Zemella Solveig Grossmann Rita Sachse Andrei Sonnabend Michael Schaefer Stefan Kubick |
author_sort |
Anne Zemella |
title |
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses |
title_short |
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses |
title_full |
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses |
title_fullStr |
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses |
title_full_unstemmed |
Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses |
title_sort |
qualifying a eukaryotic cell-free system for fluorescence based gpcr analyses |
publisher |
Nature Publishing Group |
series |
Scientific Reports |
issn |
2045-2322 |
publishDate |
2017-06-01 |
description |
Abstract Membrane proteins are key elements in cell-mediated processes. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. Up to date, there still exist a number of GPCRs that has not been structurally and functionally analyzed due to difficulties in cell-based membrane protein production. A promising approach for membrane protein synthesis and analysis has emerged during the last years and is known as cell-free protein synthesis (CFPS). Here, we describe a simply portable method to synthesize GPCRs and analyze their ligand-binding properties without the requirement of additional supplements such as liposomes or nanodiscs. This method is based on eukaryotic cell lysates containing translocationally active endogenous endoplasmic reticulum-derived microsomes where the insertion of GPCRs into biologically active membranes is supported. In this study we present CFPS in combination with fast fluorescence-based screening methods to determine the localization, orientation and ligand-binding properties of the endothelin B (ET-B) receptor upon expression in an insect-based cell-free system. To determine the functionality of the cell-free synthesized ET-B receptor, we analyzed the binding of its ligand endothelin-1 (ET-1) in a qualitative fluorescence-based assay and in a quantitative radioligand binding assay. |
url |
https://doi.org/10.1038/s41598-017-03955-8 |
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