| Autor: |
Mhurchú NN; School of Biomolecular and Biomedical Science, Conway Institute , University College Dublin , Dublin D04 V1W8 , Ireland., Zoubak L; National Institute on Alcohol Abuse and Alcoholism , National Institutes of Health , 5625 Fishers Lane , Bethesda , Maryland 20892 , United States., McGauran G; School of Biomolecular and Biomedical Science, Conway Institute , University College Dublin , Dublin D04 V1W8 , Ireland., Linse S; Department of Biochemistry and Structural Biology , Lund University , Box 118, Lund 221 00 , Sweden., Yeliseev A; National Institute on Alcohol Abuse and Alcoholism , National Institutes of Health , 5625 Fishers Lane , Bethesda , Maryland 20892 , United States., O'Connell DJ; School of Biomolecular and Biomedical Science, Conway Institute , University College Dublin , Dublin D04 V1W8 , Ireland. |
| Abstrakt: |
The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB 2 , in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study. |
