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Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor
- Source :
- Journal of structural biology. 172(1)
- Publication Year :
- 2010
-
Abstract
- G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.
- Subjects :
- Proteomics
Blotting, Western
Detergents
Fluorescence Polarization
Ligands
Binding, Competitive
Chromatography, Affinity
Cell-free system
Receptors, G-Protein-Coupled
chemistry.chemical_compound
Radioligand Assay
Structural Biology
Humans
Receptor
G protein-coupled receptor
Cell-Free System
Protein Stability
Circular Dichroism
Reproducibility of Results
Receptor, Endothelin A
Receptor–ligand kinetics
chemistry
Biochemistry
Membrane protein
Solubility
TCEP
Subjects
Details
- ISSN :
- 10958657
- Volume :
- 172
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Journal of structural biology
- Accession number :
- edsair.doi.dedup.....63d8e14a714a403389e5c620ae0f39e1