Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor.
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.
Junge
Friederike
F
Centre for Biomolecular Magnetic Resonance, Institute for Biophysical Chemistry, Goethe-University of Frankfurt/Main, Max-von-Laue-Str. 9, Frankfurt/Main, Germany.
Luh
Laura M
LM
Proverbio
Davide
D
Schäfer
Birgit
B
Abele
Rupert
R
Beyermann
Michael
M
Dötsch
Volker
V
Bernhard
Frank
F
D001667:Binding, Competitive; D015153:Blotting, Western; D002474:Cell-Free System / Q000378:metabolism*; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D005454:Fluorescence Polarization; D006801:Humans; D008024:Ligands; D055550:Protein Stability; D040901:Proteomics / Q000379:methods; D011869:Radioligand Assay; D044022:Receptor, Endothelin A / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility
D003902:Detergents; D008024:Ligands; D044022:Receptor, Endothelin A; D043562:Receptors, G-Protein-Coupled