Esf 921 serum free medium

To enable receptor expression and purification, the WT human Y₂R gene (Genewiz) was cloned into a modified pFastbac1 vector (Invitrogen) with a hemagglutinin signal sequence at the N terminus, and a PreScission protease site followed by a 10 × His-tag and a Flag tag at the C terminus. To improve protein yield and stability, two mutations H149^3.51Y and S280^6.47C were introduced into Y₂R and 28 amino acids (residues S354-V381) at the C terminus of Y₂R were truncated using standard QuikChange PCR. To facilitate crystallization, residues 2–161 of a modified T4L were fused to the receptor N terminus, and residues S251-N256 in ICL3 were replaced by residues 2–148 of a modified flavodoxin (P2A, Y98W)^{27 (link)} through overlap extension PCR. Sequences of all primers used in this work are shown in Supplementary Table 3.
High-titer recombinant baculovirus (>10⁸ viral particles per ml) of the modified Y₂R was prepared using the Bac-to-Bac Baculovirus Expression System (Invitrogen). Spodoptera frugiperda (Sf9) insect cells (Invitrogen) were grown to a density of 2 × 10⁶ cells ml⁻¹ in ESF 921 serum-free medium (Expression Systems) at 27 °C and then infected with the viral stock at a multiplicity of infection of 5. The cell pellets were harvested by centrifugation 48 h post infection and stored at −80 °C until use.

Spodoptera frugiperda 9 (Sf9) (Invitrogen) and High Five™ insect cells (Expression Systems) were cultured in ESF 921 serum-free medium (Expression Systems) at 27 °C and 120 rpm. Human embryonic kidney 293 cells containing SV40 large T-antigen (HEK293T) were cultured in DMEM (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 1 mM sodium pyruvate (Gibco) and 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO₂. Chinese hamster ovary (CHO-K1) cells were cultured in F-12 (Gibco) containing 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin at 37 °C in 5% CO₂. For cAMP and receptor expression assays, HEK293T cells were seeded into 6-well cell culture plates at a density of 7 × 10⁵ cells per well. After overnight incubation, cells were transfected with GIPR, GLP-1R or GCGR construct using Lipofectamine 2000 or Lipofectamine 3000 transfection reagent (Invitrogen). For whole-cell binding assay, CHO-K1 cells were seeded into 96-well fibronectin-treated cell culture plates at a density of 3 × 10⁴ cells per well. After overnight incubation, cells were transfected with GIPR, GLP-1R or GCGR construct using Lipofectamine 2000 transfection reagent (Invitrogen). Following 24 h culturing, the transfected cells were ready for use.

Spodoptera frugiperda (Sf9) insect cells (Expression Systems) were grown in ESF 921 serum-free medium (Expression Systems) at 27 °C and 120 r.p.m. HEK 293 T cells were purchased from the Cell Bank at the Chinese Academy of Sciences, cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum (Gibco) and maintained in a humidified chamber with 5% CO₂ at 37 °C.