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

 
: Zemella, Anne; Grossmann, Solveig; Sachse, Rita; Sonnabend, Andrei; Schäfer, Michael; Kubick, Stefan

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Scientific Reports 7 (2017), Art. 3740, 10 pp.
ISSN: 2045-2322
English
Journal Article, Electronic Publication
Fraunhofer IZI ()

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.

: http://publica.fraunhofer.de/documents/N-456253.html