A large‐scale expression strategy for multimeric extracellular protein complexes using Drosophila S2 cells and its application to the recombinant expression of heterodimeric ligand‐binding domains of taste receptor

Abstract

Many of the extracellular proteins or extracellular domains of plasma membrane proteins exist or function as homo- or heteromeric multimer protein complexes. Successful recombinant production of such proteins is often achieved by co-expression of the components using eukaryotic cells via the secretory pathway. Here we report a strategy addressing large-scale expression of hetero-multimeric extracellular domains of plasma membrane proteins and its application to the extracellular domains of a taste receptor. The target receptor consists of a heterodimer of T1r2 and T1r3 proteins, and their extracellular ligand binding domains (LBDs) are responsible for the perception of major taste substances. However, despite the functional importance, recombinant production of the heterodimeric proteins has so far been unsuccessful. We achieved the successful preparation of the heterodimeric LBD by use of Drosophila S2 cells, which have a high secretory capacity, and by the establishment of a stable high-expression clone producing both subunits at a comparable level. The method overcame the problems encountered in the conventional transient expression of the receptor protein in insect cells using baculovirus or vector lipofection, which failed in the proper heterodimer production because of the biased expression of T1r3LBD over T1r2LBD. The large-scale expression methodology reported here may serve as one of the considerable strategies for the preparation of multimeric extracellular protein complexes.

Chloride ions evoke taste sensations by binding to the extracellular ligand-binding domain of sweet/umami taste receptors

“…All protein samples used for structural and functional analyses were prepared using Drosophila S2 cells (Invitrogen) or high-expression clones for each protein sample established from S2 cells in previous studies ( Nuemket et al, 2017 ; Yamashita et al, 2017 ). No authentication or test for mycoplasma contamination was performed.…”

“…A T1r3-T105A mutation was introduced in the vector pAc_mfT1r3L ( Yamashita et al, 2017 ) by PCR. The mutant expression vector was co-introduced with pAc_mft1r2aL ( Yamashita et al, 2017 ) to Drosophila S2 cells to establish a stable high-expression clone cell as previously described ( Yamashita et al, 2017 ).…”

Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

“…The protein sample was prepared as described previously [11, 17]. Briefly, Drosophila S2 cells (Invitrogen) stably expressing C-terminal FLAG-tagged T1r2aLBD and T1r3LBD [11, 21] were cultured in ExpressFiveSFM (LifeTechnologies) for five days at 27 °C. The T1r2a/T1r3-LBD protein was purified from the culture medium by the use of ANTI-FLAG M2 Affinity Gel (SIGMA).…”

Biophysical and functional characterization of the human TAS1R2 sweet taste receptor overexpressed in a HEK293S inducible cell line

“…An interesting approach was also recently developed allowing a large-scale co-expression of TAS1R extracellular domains from Medaka fish (mf). The two subunits mfTAS1R2-VFT/mfTAS1R3-VFT were expressed and purified in a heterodimeric state using Drosophila S2 cells 31 . The high expression of both subunits enabled biophysical and structural analyses such as isothermal calorimetry and small-angle X-ray scattering for the detection of ligand binding and conformational change upon taste substance binding 32 , and structure resolution by crystallization 14 .…”