GPCR Purification
Creative Biolabs provides professional services for GPCR purification to support and facilitate the cutting-edge programs of GPCR research and drug development.
Introduction of GPCR Purification
G protein-coupled receptors (GPCRs), with more than 800 members, constitute the largest family of membrane receptors. They are central players in responding to a wide variety of extracellular stimuli and mediate diverse cellular responses. GPCRs, which are involved in the pathogenesis of numerous diseases, are of particular importance for drug discovery.
GPCRs, as integral membrane proteins, are generally unstable when removed from their membrane environment. This makes it difficult to apply the wide range of biophysical and structural techniques for soluble proteins to GPCRs. After solubilization, GPCRs always need some robust purification protocol prior to further studies. Optimization of purification is still a matter of trail-and-error. Throughout the purification procedure, the most important thing is to ensure that the experimental conditions can maintain the active state of GPCRs. General affinity tags, receptor-specific ligand column, and size exclusion chromatography are commonly used for GPCR purification.
Figure 1. Schematic illustration of the affinity chromatographic purification of a target membrane protein expressed in a bacterial host. (Perez C & Maier T., 2020)
Our Services
We have established an unparalleled platform for GPCR purification to provide the most reliable services for various projects. Whether you need labeled or untagged GPCRs, single protein or GPCR complex, small-scale or large-scale purification, our skillful and experienced scientists can develop the optimal strategies to meet your specific requirements.
Affinity chromatography is commonly used as the first chromatographic step in GPCR purification. Recombinant cloning techniques make it easy to introduce general affinity tags at the N-terminal or C-terminal of GPCRs. Receptor-specific ligand affinity chromatography may be required as the second step to isolate pure and functional GPCRs.
Ion-exchange chromatography is not a general purification method, but can be used in the purification of several GPCRs. Anion-exchange chromatography is often utilized for GPCR purification. Anionic detergents with anion-exchange columns and cationic detergents with cation-exchange columns need to be avoided.
Size-exclusion chromatography (SEC), also known as molecular sieve chromatography, is an important purification step before structural studies and can be used as a final step before in surfo crystallization of several GPCRs.
Although affinity tags are very useful in GPCR purification, they may interfere with the GPCR functions and structural studies. Proteolytic cleavage can be used to remove these flexible regions, which might also produce intense and overlapping NMR signals.
Glycosylation is important for proper folding and functionality of GPCRs. However, glycan heterogeneity and flexibility might prevent formation of GPCR crystals. Deglycosylation is a generally preferred method to prepare purified GPCR samples for crystallization.
Evaluation of Membrane Protein
The purified GPCRs will be evaluated carefully using SDS-PAGE to ensure the expected properties of the target proteins can be maintained. It is quite important for further functional and structural studies.
Creative Biolabs is always ready to provide the best services to each of our customers. If you are interested in our services or have any specific requirements, please feel free to contact us for more details.
References
- Perez C & Maier T. Expression, Purification, and Structural Biology of Membrane Proteins. 2020.
- Grisshammer R. Purification of recombinant G protein-coupled receptors. Methods in enzymology. Academic Press, 2009, 463: 631-645.
- Milić D & Veprintsev D B. Large-scale production and protein engineering of G protein-coupled receptors for structural studies. Frontiers in pharmacology, 2015, 6: 66.