Unit

UNIT 29.4 Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris

  1. Shweta Singh1,
  2. Minghao Zhang1,
  3. Nicolas Bertheleme1,
  4. Philip G. Strange2,
  5. Bernadette Byrne1

Published Online: 1 FEB 2012

DOI: 10.1002/0471140864.ps2904s67

Current Protocols in Protein Science

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.

Author Information

  1. 1

    Membrane Protein Crystallography Group, Division of Molecular Biosciences, Imperial College London, United Kingdom

  2. 2

    School of Pharmacy, University of Reading, Whiteknights, Reading, United Kingdom

Publication History

  1. Published Online: 1 FEB 2012
  2. Published Print: FEB 2012

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 (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.

Keywords:

  • G protein−coupled receptor;
  • adenosine A2aR;
  • immobilized metal affinity chromatography;
  • size-exclusion chromatography;
  • ion exchange;
  • antibody affinity chromatography;
  • SDS-PAGE;
  • radioligand binding analysis;
  • aggregation