Search

Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein   

Download PDF How to cite Favorites Q&A Share your feedback Cited by

In this protocol

Original research article

A brief version of this protocol appeared in:
Nature
Aug 2016

Abstract

G protein-coupled receptors (GPCRs) promote cytoplasmic signalling by activating heterotrimeric G proteins in response to extracellular stimuli such as light, hormones and nucleosides. Structure determination of GPCR–G protein complexes is central to understanding the precise mechanism of signal transduction. However, these complexes are challenging targets for structural studies due to their conformationally dynamic and inherently transient nature. We recently developed an engineered G protein, mini-Gs, which addressed these problems and allowed the formation of a stable GPCR–G protein complex. Mini-Gs facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its G protein-bound conformation at 3.4 Å resolution. Here, we describe a step by step protocol for the expression and purification of A2AR, and crystallisation of the A2AR–mini-Gs complex.

Keywords: Adenosine A2A receptor, A2AR, Active state, GPCR, G protein-coupled receptor, Mini G protein, Mini-Gs, G protein complex

Background

We recently developed an engineered minimal G protein, mini-Gs (Carpenter and Tate, 2016), which facilitated the structure determination of the human adenosine A2A receptor (A2AR) in its active state (Carpenter et al., 2016). Mini-Gs stabilises the active conformation of A2AR sufficiently to allow crystallization of the complex by vapour diffusion in the detergent octylthioglucoside. Here, we describe a detailed protocol for the expression and purification of A2AR, which is adapted from a previously described method developed in our laboratory (Lebon et al., 2011a and 2011b; Tate and Lebon, 2015). We also describe a step by step procedure for the preparation and crystallisation of the A2AR–mini-Gs complex, earlier described in Carpenter et al. (2016). Expression and purification of mini-Gs is described in a companion manuscript (Carpenter and Tate, 2017).

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Carpenter, B. and Tate, C. G. (2017). Expression, Purification and Crystallisation of the Adenosine A2A Receptor Bound to an Engineered Mini G Protein. Bio-protocol 7(8): e2234. DOI: 10.21769/BioProtoc.2234.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.