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Expression and Purification of Mini G Proteins from Escherichia coli   

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Original research article

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

Abstract

Heterotrimeric G proteins modulate intracellular signalling by transducing information from cell surface G protein-coupled receptors (GPCRs) to cytoplasmic effector proteins. Structural and functional characterisation of GPCR–G protein complexes is important to fully decipher the mechanism of signal transduction. However, native G proteins are unstable and conformationally dynamic when coupled to a receptor. We therefore developed an engineered minimal G protein, mini-Gs, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A2A receptor (A2AR) in its active conformation. Mini G proteins are potentially useful tools in a variety of applications, including characterising GPCR pharmacology, binding affinity and kinetic experiments, agonist drug discovery, and structure determination of GPCR–G protein complexes. Here, we describe a detailed protocol for the expression and purification of mini-Gs.

Keywords: Complex, Engineered G protein, G protein-coupled receptor, GPCR, Mini G protein, Mini-Gs

Background

We recently reported the development of an engineered minimal G protein, mini-Gs (Carpenter and Tate, 2016), which facilitated the crystallisation of the human adenosine A2A receptor (A2AR) in its active conformation (Carpenter et al., 2016; Carpenter and Tate, 2017). Unlike heterotrimeric G proteins, which require expression in eukaryotic systems, mini-Gs is highly expressed in Escherichia coli (E. coli) and can be easily purified with a yield of 50-100 mg of mini-Gs per liter of culture. Here, we describe a step by step protocol, earlier described in Carpenter and Tate (2016), that can be used for the expression and purification of any of the mini G protein constructs described previously (Carpenter et al., 2016; Carpenter and Tate, 2016). Since mini-Gs construct 393 is well suited to most applications (see Carpenter and Tate, 2016), it will be used as an example herein.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Carpenter, B. and Tate, C. G. (2017). Expression and Purification of Mini G Proteins from Escherichia coli. Bio-protocol 7(8): e2235. DOI: 10.21769/BioProtoc.2235.
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