Purification of the human G protein-coupled receptor adenosine A(2a)R in a stable and functional form expressed in
Shweta Singh1, Minghao Zhang1, Nicolas Bertheleme1
1Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom.
Abstract:
The isolation of membrane proteins with the aim of producing highly pure, homogeneous, stable, and functional material remains challenging, and it is often necessary to develop protein-specific purification protocols by trial and error. One key tool that is required in the development of a suitable protocol is a functional assay. This unit describes a range of different protocols for isolation of the human adenosine A2a receptor (A(2a)R). These protocols show the importance of developing a robust method for comparing the quality of protein obtained by a combination of biophysical analyses including SDS-PAGE, analytical size-exclusion chromatography, and functional analysis. One of the keys to isolating and maintaining a functional receptor, found not only in the optimal protocol described here but in other published examples, is that there should be no more than two chromatographic steps.
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