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.
Highlights
G protein-coupled receptors (GPCRs) are known to form the largest class of transmembrane proteins in humans
To illustrate the function of GPCRs synthesized in an insect-based cell-free system, the Endothelin B receptor was chosen as a model protein
The first part of this report is focused on the localization, integration and orientation of synthesized endothelin B (ET-B) receptor into microsomal membranes since its correct folding and functionality requires a proper membrane insertion[16]
Summary
G protein-coupled receptors (GPCRs) are known to form the largest class of transmembrane proteins in humans. In a current report based on an E. coli cell-free system, parts of the Sec translocon machinery were reconstituted inside of cell-sized liposomes during the synthesis of the target protein[8]. With this modification the productivity of the system was significantly increased. The presence of a functional translocon complex might be a notable advantage for membrane protein synthesis and analysis in eukaryotic cell-free systems harboring endogenous microsomal structures. Eukaryotic lysates from cultured insect (Sf21) and Chinese hamster ovary cells (CHO) contain endogenous microsomal structures with an integrated natural translocon[13, 14] The productivity of these systems is currently lower in comparison to E. coli and wheat germ systems. A quantitative measurement of ligand affinity revealed a typical ET-1 binding curve, thereby showing the correct binding parameters of the cell-free produced ET-B receptor
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