Ability to regenerate/re-use the matrixĬurrently, there only a few examples of commercial kits that satisfy these requirements.Ability to elute the captured protein in mild buffer conditions.High affinity in the presence of detergents (ideally in the picomolar or low nanomolar range).When choosing the tag/affinity matrix pair for purification of membrane proteins for structural studies, the following parameters should be considered: Several other affinity tag/matrix pairs have been described in the literature, but very few of them have been applied successfully to preparative-scale production of GPCR. 3,16 However, the application of FLAG resin for preparation of multi-milligram quantities of GPCR is expensive, especially in the case of commercial preparation of affinity resins. The antibody-epitope interaction exploited by such systems as FLAG-tag/ FLAG resin may produce a high purity of the target protein, even in the presence of detergents. However, IMAC is not always efficient for purification of GPCRs, especially for those targets that are expressed at low levels, because of the low affinity of interaction between the His-tag and the resin in detergent containing buffers. Plus, get special offersįrom American Pharmaceutical Review – all delivered right to your inbox! Sign up now! Stay up to date with the latest news, articles, and events. Poly-histidine (immobilized-metal affinity chromatography, IMAC) and FLAG tags have been successfully used. Purification of a recombinant GPCR typically involves one or more steps of affinity chromatography, relying on engineered tag(s) fused to the expressed protein. 6-8 Structural studies can provide critical contributions to the rational design of novel, specific drugs targeting this receptor. Human cannabinoid receptor CB2 is primarily expressed in cells of immune origin, and is an attractive target for the development of drugs for management of inflammation, immunological disorders and pain. In recent years, significant improvements have been made in designing efficient protocols for expression of GPCRs in different cell types, their stabilization, and purification by affinity chromatography.
This is notoriously challenging, because of the relatively low expression levels of GPCR in membranes and their instability in detergents. Purification of GPCRs typically requires solubilization of these receptors from cell membranes into detergent micelles, followed by one or more steps of affinity purification. The high resolution structural studies require large quantities of pure, homogenous and stable proteins. G protein-coupled receptors (GPCR) comprise a large family of integral membrane proteins involved in a wide array of cell signaling pathways. It is feasible that similar strategies can be successfully employed for expression and purification of other membrane protein targets. This technique was successfully applied to the purification of the recombinant cannabinoid receptor CB2, a promising target for the development of drugs for inflammation, immunological disorders and pain. Here, we describe a recently introduced purification system employing a high affinity molecular switch based on fragment complementation, with a calcium dependent capture and EDTA mediated chelation elution. Because of relatively low expression levels of these recombinant receptors, it is challenging to design an efficient strategy for selective and efficient purification with high yield. Purification of recombinant GPCRs typically involves their solubilization into detergent micelles followed by chromatographic purification. For high resolution structural studies of these receptors, multi-milligram quantities of pure and structurally unperturbed proteins are required. Heptahelical G protein-coupled receptors (GPCR) comprise a large family of integral membrane proteins involved in a wide array of cell signaling pathways. School of Biomolecular and Biomedical Science, Conway Institute
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