GFP‐based evaluation system of recombinant expression through the secretory pathway in insect cells and its application to the extracellular domains of class C GPCRs

Abstract

Applications of the GFP-fusion technique have greatly facilitated evaluations of the amounts and qualities of sample proteins used for structural analyses. In this study, we applied the GFP-based sample evaluation to secreted protein expression by insect cells. We verified that a GFP variant, GFPuv, retains proper folding and monodispersity within all expression spaces in Sf9 cells, such as the cytosol, organelles, and even the extracellular space after secretion, and thus can serve as a proper folding reporter for recombinant proteins. We then applied the GFPuv-based system to the extracellular domains of class C G-protein coupled receptors (GPCRs) and examined their localization, folding, and oligomerization upon insect cell expression. The extracellular domain of metabotropic glutamate receptor 1 (mGluR1) exhibited good secreted expression by Sf9 cells, and the secreted proteins formed dimer with a monodisperse hydrodynamic state favorable for crystallization, consistent with the results from previous successful structural analyses. In contrast, the extracellular domains of sweet/umami taste receptors (T1R) almost completely remained in the cell. Notably, the T1R and mGluR1 subfractions that remained in the cellular space showed polydisperse hydrodynamic states with large aggregated fractions, without forming dimers. These results indicated that the proper folding and oligomerization of the extracellular domains of the class C GPCR are achieved through the secretory pathway.

Structural architecture of a dimeric class C GPCR based on co-trafficking of sweet taste receptor subunits

“…In this study, we identify a conserved surface during in vitro selection of human T1R2 for associating with T1R3. Identifying the dimerization surface of the sweet taste receptor was challenging due to low surface expression (2,38,39), which necessitated the selection of T1R2 mutants for enhanced surface expression and co-trafficking with T1R3. A subsequent deep mutational scan of T1R2 in human cells identified surfaces on lobe 1 of the LBD, on the CRD, and in TM6 for dimerization with T1R3 as assessed by their co-trafficking to the plasma membrane.…”

Taste substance binding elicits conformational change of taste receptor T1r heterodimer extracellular domains

“…So far, not only the structural analyses but also the molecular functional analyses using the purified protein of T1rs have been completely hampered, due to the difficulties in the heterologous expression and purification even for the partial regions such as the LBD 19 . In this study, we found that the LBDs of T1r2 and T1r3 of medaka fish ( Oryzias latipes , also known as Japanese rice fish), a representative model organism in vertebrates, can be expressed heterologously as a properly folded and functional heterodimeric protein, thus enabling various biophysical analyses.…”

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

“…We previously found that T1r2aLBD and T1r3LBD from medaka fish displayed favorable secreted expression and stable heterodimerization, although those from other sources, including mouse and human, displayed failure of folding and localization during the recombinant expression . To address the large‐scale preparation of the heterodimeric T1rLBD, we first attempted baculovirus expression, the most commonly used eukaryotic expression system for structural studies.…”