Expression, purification, refolding and structure analysis of stabilized G protein-coupled receptors expressed in E. coli inclusion bodies
FacultiesFakultät für Naturwissenschaften
InstitutionsKompetenzzentrum "Ulm Peptide Pharmaceuticals (U-PEP)"
External cooperationsHochschule Biberach
G protein–coupled receptors are membrane proteins involved in signal transduction across the biological lipid bilayer. This receptor class represent both the largest membrane protein and drug target family, accounting for ~800 members encoded in the human genome. GPCRs are distributed across nearly all of the body’s organs and tissues, which makes them a key regulatory element in a broad range of normal and pathological processes. The structure determination of G protein-coupled receptors with high-resolution crystals is a key focus in membrane protein biology to understand the basic mechanism of their activation and signaling for structure-based drug design. High-resolution crystal structures in combination with multiple complex structures for a specific target are a prerequisite for a successful structure-based drug discovery. Currently, obtaining X-ray structures of membrane proteins is still far from becoming routine due to their notorious instability in detergent micelles, the inherent conformational flexibility, bottlenecks in overexpressing large quantities of the recombinant receptor and the hydrophobic protein surface, which often impairs crystallization. In the present project, we overexpressed two G protein-coupled receptors in Escherichia coli inclusion bodies. One of these two receptors is the sphingosine-1-phosphate receptor 1 with known structure, which was used for validation purposes. The second receptor was the orphan G protein-coupled receptor 3 with unknown structure. Both receptors were refolded in vitro by detergent exchange into detergent micelles. The monomeric form of the refolded receptors was isolated by size exclusion chromatography and quality checked using a thermal stability assay. Receptor stability was optimized for higher thermal stability through improving refolding conditions into mixed lipid detergent micelles. Ligand binding was shown for both receptors, which were crystallized in the lipidic cubic phase. Beside crystal formation for both receptors in different crystallization conditions, the reproduction of sphingosine-1-phosphate receptor 1 crystallization conditions resulted in crystal formation. Harvested crystals were analyzed at a synchrotron beamline, which resulted in non-diffracting potential protein crystals for both receptors.
Subject HeadingsG-Protein gekoppelter Rezeptor [GND]
Escherichia coli [LCSH]