Guanine nucleotide-binding proteins (G proteins) are an essential component in G-protein coupled receptor (GPCR) mediated signaling. They are stimulated by a ligand-induced conformational change in the receptor, which catalyzes the exchange of GDP, bound to the ‘resting’ state of the Gα subunit, with GTP. It is thought that the GTP-bound G protein dissociates into two signaling entities: the Gα subunit bound to GTP and the Gβγ subunit, both of which activate downstream effectors to initiate a physiological response. That response is then terminated when the Gα subunit hydrolyzes GTP to GDP and re-associates with the Gβγ subunit, and the process can begin anew [reviewed by 1]. The α subunit is the primary determinant for which receptor the G protein couples and the defining feature of the G protein. Therefore, G proteins are categorized by the Gα subunit. Together they make up four subfamilies: the Gs subfamily stimulates adenylyl cyclase [2]; the Gi subfamily inhibits adenylyl cyclase and regulates ion channels [3, 4]; the Gq/11 subfamily activates phospholipase C beta and results in an influx of Ca2+ [5]; and the G12/13 subfamily regulates small GTP binding proteins, such as rho, which modulates the cytoskeleton [6]. Approximately twenty different subtypes of the α subunit exist [7]. Five subtypes of the β subunit [8] and 11 subtypes of the γ subunit [9, 10] have been identified as well. Combinations of the different α, β and γ subtypes provide a great diversity of signaling pathways that can be regulated by GPCRs, other activators of G protein signaling (AGS) or regulators of G protein signaling (RGS). It is interesting to note that very little is known about how GPCRs selectively couple to a single subtype of the α subunit. This phenomenon is especially striking considering there are a thousand GPCRs of diverse primary structure [11], yet there are only four subfamilies of G proteins. High-resolution structural insight into the receptor- G protein complex is difficult to obtain due to the dynamic nature of the interaction. Currently, static images of the various states of the Gα subunit [12–16], the inactive structure of rhodopsin [17] and recently the structure of Giα in the presence of ICL3 receptor mimic and βγ-interacting peptides [18] have been described. These images, along with biophysical studies [19–21], have supplied knowledge about the receptor- G protein interaction, but a complete depiction of the ternary complex is still elusive [22]. The G protein interacts with many non-GPCR related proteins, or activators of G protein signaling (AGS), that share no sequence homology and activate G proteins by different signaling mechanisms [23]. In order to understand the differential activation and the fluctuation of G protein states, a comprehensive collection of G protein structures needs to be obtained [1]. Heterologous expression systems have been successful at generating the copious amount of isotope-labeled soluble protein needed for high-resolution NMR studies in other protein systems. As a step towards these structural studies of G protein states, we need to utilize and improve the use of Escherichia coli (E. coli), which can provide a scalable production source of recombinant proteins. Advantages include speed, inducibility for control of optimal expression, isotope labeling for high-resolution NMR studies, cost and ease of purification. Of the twenty different subtypes of the Gα subunit identified, five have been successfully expressed in E. coli: wild-type and mutant forms of Gsα, Gi1α, Gi2α, Gi3α and Goα, all of which only differ from their mammalian sources by post-translational covalent modification. However, the expression of these proteins is not without its challenges. The current protocol requires long growth times (24–36 h) at moderate temperature (30 °C) and often multi-step purification to obtain active protein [24]. Expression levels of active protein vary depending on the α subunit expressed; Gsα expression typically yields 0.1–1 mg/L, while Giα expression yields 40 mg/L [24, 25]. Many of the Gα subunits (Gqα family, G12α family and Gtα (transducin) from the Giα family) have not been successfully obtained from bacterial systems. Manipulation at the DNA level to produce chimeras of different α subunits has been successful at producing proteins that resemble these difficult-to-express proteins: a transducin/Giα chimera was made to achieve soluble active transducin-like protein, expressed in E. coli, at levels of 3–6 mg/L for biophysical studies [26, 27]. Chimeras of G12α and G13α, also with Giα, were used to produce these hard-to-express Gα subunits in baculovirus systems [28]. Interestingly these chimeras only differ from their parent protein by 11 amino acids. The 11 amino acid exchange allowed for difficult-to-express Gα production in the recombinant systems while maintaining the parent Gα selectivity. Table 1 summarizes the literature available on the current expression systems used to acquire the representative members of the Gα subunits for structural characterization. TABLE 1 Previously reported expression yields for representative Gα subunits. While Sf9 cells have been successful at producing some of the more challenging Gα subunits they have several drawbacks. Recombinant protein production from insect cells is time-consuming and laborious and often gives rise to heterogeneous populations of proteins, due to variable posttranslational modifications. Sf9 cells also natively express certain G alpha subunits – Gsα, Goα and Gqα– which account for 10–40% of total active Gα subunit. These contaminating endogenous Gα subunit require additional purification procedures, with final yields in the μg/L range [29]. Methods for expressing the protein in inclusion bodies and then efficiently refolding the Gα subunits would be of great value for characterization of the structure and properties of the Gα subunit. In addition, Gα is quite prone to loss of activity upon storage at 4 °C, regardless of the original source of expression, requiring storage in small samples at −80 °C in order to prevent denaturation. A refolding method would allow for E. coli production of Gα subunits that can be stored in denaturant at room temperature or as inclusion bodies and quickly refolded when needed. Successfully refolding Gα would enable a rescue of the protein lost during expression, a simplified method of production for those Gα that have proven recalcitrant to other production methods, and sufficient quantities of isotope-labeled Gα subunits for high-resolution NMR studies.