Expression and Purification of Mini G Proteins from Escherichia coli

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-G_s, which formed a stable complex with GPCRs, and facilitated the crystallisation and structure determination of the human adenosine A_2A receptor (A_2AR) 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-G_s.

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

Figure 1.. Mini-G_s393 E. coli expression construct.

Mini-G_s was cloned into the plasmid pET15b (the sequence and map of this vector are available from the EMD Millipore website) using NcoI and XhoI restriction sites (underlined). The Mini-G_s393 construct contains an N-terminal 6x histidine tag (highlighted in red) followed by a TEV protease cleavage site (highlighted in green). Start and stop codons are shown in bold.

Figure 2.. SDS-PAGE and gel filtration analysis of the mini-G_s393 purification.

A. SDS-PAGE analysis of the mini-G_s393 purification. Lane: (M) molecular weight markers; (1) HisTrap column loading material (1:10 dilution); (2) HisTrap column flow through (1:10 dilution); (3) HisTrap column wash; (4) HisTrap column eluate; (5) sample after TEV digestion; (6) Ni²⁺-NTA negative purification flow through; (7) Ni²⁺-NTA negative purification eluate; (8) gel filtration pool. TEV protease is indicated by an asterisk. Note that it is not necessary to boil the samples before loading on the gel. B. Preparative gel filtration chromatogram for mini-G_s393. Pooled fractions are indicated by dashed lines, fractions at the leading edge of the peak should be omitted to ensure that aggregates are completely removed. Note that the UV detector saturates at absorbance readings above 2,700 mAu, which gives the chromatogram a truncated absorbance profile. C. Analytical gel filtration analysis of purified mini-G_s on a Superdex 200 GL 10/300 column. The chromatogram demonstrates that purified mini-G_s is monomeric (calculated molecular weight of 23 kDa compared to the theoretical value of 27 kDa) and free from aggregates.