An efficient Escherichia coli expression system for the production of a functional N-terminal domain of the T1R3 taste receptor

Abstract

Sweet taste is mediated by a dimeric receptor composed of two distinct subunits, T1R2 and T1R3, whereas the T1R1/T1R3 receptor is involved in umami taste perception. The T1R1, T1R2, and T1R3 subunits are members of the small family of class C G protein-coupled receptors (GPCRs). The members of this family are characterized by a large N-terminal domain (NTD), which is structurally similar to bacterial periplasmic-binding proteins and contains the primary ligand-binding site. In a recent study, we described a strategy to produce a functional dimeric human T1R3-NTD. Although the protein was expressed as inclusion bodies (IBs) using the Escherichia coli system, the conditions for the refolding of functional hT1R3-NTD were determined using a fractional factorial screen coupled to a binding assay. Here, we report that this refolding strategy can be used to produce T1R1- and T1R2-NTDs in large quantities. We also discuss that our findings could be more generally applicable to other class C GPCR-NTDs, including the γ-aminobutyric acid type B receptor (GABABR), the extracellular calcium-sensing receptor (CaSR) and the large family of pheromone (V2R) orphan receptors.

Keywords: Escherichia coli; GPCR; bacteria; expression; recombinant protein; sugar; sweet receptor; sweetener; taste; umami receptor.

Figure 1. Expression of T1R-NTD proteins. (A) The NTDs of the T1R1, T1R2 and T1R3 proteins, minus a putative signal peptide and without the CRR, were expressed independently of the seven-transmembrane domain. (B) The construct pET28-hT1R3-NTD encodes a protein comprising an N-terminal His6-tag that can be cleaved by thrombin, followed by hT1R3-NTD and a C-terminal Strep-tag II. (C) The pET28-hT1R1-NTD/pET28-hT1R2-NTD plasmid encodes a fusion protein that contains an N-terminal His6-tag that can be cleaved with thrombin, followed by hT1R1- or hT1R2-NTD and a C-terminal His6-tag to facilitate the purification of the heterodimers. (D) The pET22-hT1R3-NTD plasmid encodes hT1R3-NTD with an additional N-terminal Met residue and a C-terminal Strep-tag II. The thrombin cleavage site is represented with an arrow.

Figure 2. Purified inclusion bodies of hT1R-NTDs expressed using the pET28 vector. The proteins were separated by 12% SDS-PAGE and stained with Coomassie blue. The molecular mass markers are in lane M.

Figure 3. SDS-PAGE analysis of hT1R3-NTD expressed in E. coli BL21 (DE3) cells transformed with pET22-hT1R3-NTD (lane 1) and pET28-hT1R3-NTD (lane 2). The position of hT1R3-NTD is indicated with an arrow. Protein expression was induced with 1 mM IPTG for 3 h. The cell lysates were loaded onto 12% gels for SDS-PAGE, with molecular mass markers (lane M). The gel was stained with Coomassie blue.

Figure 4. Outline of the strategy for the production of hT1R3-NTD.