UNIT 29.4 Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris
Published Online: 1 FEB 2012
Copyright © 2011 by John Wiley & Sons, Inc.
Lab Protocol Title
Current Protocols in Protein Science
How to Cite
Singh, S., Zhang, M., Bertheleme, N., Strange, P. G. and Byrne, B. 2012. Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris. Current Protocols in Protein Science. 29:29.4:29.4.1–29.4.17.
- Published Online: 1 FEB 2012
- Published Print: FEB 2012
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 (A2aR). 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.
- G protein−coupled receptor;
- adenosine A2aR;
- immobilized metal affinity chromatography;
- size-exclusion chromatography;
- ion exchange;
- antibody affinity chromatography;
- radioligand binding analysis;