Human Y2 Receptor Expression and Purification

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.