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.

Figure 1

Transient expression of GFPuv in Sf9 cells, using the vectors for cytosolic expression (A, C, E) and secreted expression (B, D, F). (A, B) Western-blotting analyses of the expression products from Sf9 cells transfected with the pCGFP_SF vector (cytosolic expression, panel A) or the pCGFP_SF+AKH vector (secreted expression, panel B) at 120 h after transfection. Lanes marked “C” and “S” show cellular (cytoplasm and organelles) and secreted fractions, respectively. (C, D) FSEC elution profiles of the samples transfected with the pCGFP_SF vector (C) or the pCGFP_SF+AKH vector (D). The same samples used in panels A and B were subjected to the analyses. Cyan and red lines show the profiles of the cellular (cytoplasm and organelles) and secreted fractions, respectively. The inset shows a magnification of the profile of the cellular fraction. (E, F) Confocal microscopic observation of Sf9 cells transfected with the pCGFP_SF vector (E) or the pCGFP_SF+AKH vector (F) at 72 h after transfection. The scale bars represent 5 μm.

Figure 2

Quantitative analyses of recombinant GFPuv in transiently transfected Sf9 cells, at each time point after transfection. Line graphs represent relative fluorescence intensity measured by in vivo fluorescence detection with a microplate reader, while cyan and red bars describe the amounts of GFPuv in the cellular (cytoplasm and organelles) and secreted fractions of the same culture samples evaluated by FSEC, respectively. (A) Cytosolic expression of GFPuv (B) secreted expression of GFPuv with the AKH signal sequence. The error bars for each data point represent the standard deviation from triplicate measurements.

Figure 3

Expression analyses of mGluR1LBD-GFPuv by Sf9 cells at 120 h after transfection. (A) Western blotting analysis. Lanes marked “C” and “S” show cellular (cytoplasm and organelles) and secreted fractions, respectively. (B) FSEC profiles. Cyan and red lines show the elution profiles of the cellular (cytoplasm and organelles) and secreted fractions, respectively. The closed and open triangles represent the elution volumes corresponding to dimeric and monomeric mGluR1LBD-GFPuv, respectively. The inset shows a magnification of the cellular fraction profile. (C) Typical observations of Sf9 cells expressing mGluR1LBD-GFPuv by confocal-microscopy at 72 h after transfection. The scale bar represents 5 μm.

Figure 4

Expression analyses of T1RsLBD-GFPuv by Sf9 cells at 72 h after transfection. (A–G) Expression of each T1RsLBD-GFPuv gene. (H–L) Coexpression of heterodimeric pairs of T1RsLBD-GFPuv genes. (A, H) Western blotting analyses. Lanes marked “C” and “S” show the cellular (cytoplasm and organelles) and secreted fractions, respectively, and the numbers above each lane correspond to the samples as follows: 1, T1R1; 2, T1R2; 3, T1R3; 1/3, coexpression of T1R1 and T1R3; 2/3, coexpression of T1R2 and T1R3. (B–D) and (I, J), FSEC elution profiles. Cyan and red lines show the cellular and secreted fractions, respectively. The open triangle represents the elution volume of the monomeric T1RsLBD-GFPuv. (B) T1R1; (C) T1R2; (D) T1R3; (I) coexpression of T1R1 and T1R3; (J) coexpression of T1R2 and T1R3. (E–G), (K, L) Typical confocal microscopic observations of T1RsLBD-GFPuv expressing Sf9 cells. (E) T1R1; (F) T1R2; (G) T1R3; (K) coexpression of T1R1 and T1R3; (L) coexpression of T1R2 and T1R3. The scale bars represent 5 μm.

Figure 5

Expression analyses of AKHss-T1RsLBD-GFPuv by Sf9 cells at 72 h after transfection. (A–G) Expression of each AKHss-T1RsLBD-GFPuv gene. (H–L) Coexpression of heterodimeric pairs of AKHss-T1RsLBD-GFPuv genes. (A, H) Western blotting analyses. Lanes marked “C” and “S” show the cellular (cytoplasm and organelles) and secreted fractions, respectively, and the numbers above each lane correspond to the samples as follows: (1) T1R1; (2) T1R2; (3) T1R3; 1/3, coexpression of T1R1 and T1R3; 2/3, coexpression of T1R2 and T1R3. (B–D), (I, J) FSEC elution profiles. Cyan and red lines show the cellular and secreted fractions, respectively. The open triangle represents the elution volume of the monomeric AKHss-T1RsLBD-GFPuv. (B) T1R1; (C) T1R2; (D) T1R3; (I) coexpression of T1R1 and T1R3; (J) coexpression of T1R2 and T1R3. (E–G), (K, L) Typical confocal microscopic observations of AKHss-T1RsLBD-GFPuv expessing Sf9 cells. (E) T1R1; (F) T1R2; (G) T1R3; (K) coexpression of T1R1 and T1R3; (L) coexpression of T1R2 and T1R3. The scale bars represent 5 μm.