Expi293f inducible human embryonic kidney cells

The NK1R-miniG_s/q70 fusion construct (generation described above) was modified to replace the miniG_s/q70 protein with the miniG_s399²¹ protein using Gibson cloning. The subsequent NK1R-miniG_s399 fusion construct was transiently transfected into 200-mLs of Expi293F^™ Inducible Human Embryonic Kidney Cells (unauthenticated and untested for mycoplasma contamination, Life Technologies) using the Expifectamine Transfection Kit (Life Technologies), following the manufacturer’s instructions. Expression of the NK1R-miniG_s399 fusion protein was induced and enhanced 18 hours after transfection with addition of 1 μg/mL doxycycline hyclate (Sigma Aldrich), 10 mM sodium butyrate (Sigma Aldrich), and addition of enhancers from the Expifectamine Transfection Kit, as per the manufacturer’s instructions. Cells were harvested 24 hours after induction and stored at −80 °C until further use.
The SP-NK1R-miniG_s399 fusion protein was purified exactly as described above for the SP-NK1R-miniG_s/q70 fusion protein. Incubation of SP-NK1R-miniG_s399 fusion protein with Gβ₁γ₂ and Nb35 was performed exactly as described above for the SP-NK1R-miniG_s/q70 complex. The resulting SP-NK1R-miniG_s399 heterotrimeric complex was concentrated with a 50 kDa MWCO spin concentrator to 2.86 mg/mL (20 μM) for preparation of cryo electron microscopy grids.