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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Autores principales:	Anne Zemella, Solveig Grossmann, Rita Sachse, Andrei Sonnabend, Michael Schaefer, Stefan Kubick
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5c74a40789144049b2f636de8b2e6d9
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spelling	oai:doaj.org-article:a5c74a40789144049b2f636de8b2e6d92021-12-02T11:52:17ZQualifying a eukaryotic cell-free system for fluorescence based GPCR analyses10.1038/s41598-017-03955-82045-2322https://doaj.org/article/a5c74a40789144049b2f636de8b2e6d92017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03955-8https://doaj.org/toc/2045-2322Abstract 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.Anne ZemellaSolveig GrossmannRita SachseAndrei SonnabendMichael SchaeferStefan KubickNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Anne Zemella Solveig Grossmann Rita Sachse Andrei Sonnabend Michael Schaefer Stefan Kubick Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses
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.
format	article
author	Anne Zemella Solveig Grossmann Rita Sachse Andrei Sonnabend Michael Schaefer Stefan Kubick
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 Portfolio
publishDate	2017
url	https://doaj.org/article/a5c74a40789144049b2f636de8b2e6d9
work_keys_str_mv	AT annezemella qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses AT solveiggrossmann qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses AT ritasachse qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses AT andreisonnabend qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses AT michaelschaefer qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses AT stefankubick qualifyingaeukaryoticcellfreesystemforfluorescencebasedgpcranalyses
_version_	1718395122469568512

Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses

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