Expifectamine transfection kit

The heavy and light chain sequences of mAb45D8^{23 (link)} were separately cloned into pcDNA3.4 and the resulting vectors were transfected into Expi293F Human Embryonic Kidney cells (unauthenticated and untested for mycoplasma contamination, Life Technologies) using a 1:2 mass ratio of light and heavy chain DNA with the Expifectamine transfection kit (Life Technologies) as per the manufacturer’s instructions. Supernatant containing mAb45 was harvested 136 h after transfection and loaded on homemade Protein G Sepharose beads and extensively washed with a buffer comprising 20 mM HEPES pH 7.50, and 100 mM NaCl. mAb45D8 was eluted with 100 mM glycine (pH 3.0) and fractions were immediately neutralized with 200 mM HEPES pH 7.50. To generate the Fab fragment, 10 mg of purified mAb45D8.1 was diluted into 9.5 ml freshly prepared cleavage buffer (20 mM sodium phosphate pH 7.00, 10 mM EDTA, and 10 mM cysteine) and treated with 0.5 ml agarose immobilized papain (Thermo Scientific) at 37°C. After 16 h the cleaved Fab45D8 fragment was purified by reverse Protein A affinity chromatography, followed by SEC into buffer comprising 20 mM HEPES pH 7.50 and 100 mM NaCl. Fab45D8 was concentrated on a Vivaspin 10-kDa MWCO concentrator, and aliquots were flash frozen in liquid nitrogen and stored at −80°C for later use.

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.

The sequence of the SARS-CoV-2 RBD gene (amino acids 319–541) was synthesized by GenScript Co., Ltd., and cloned into the pCAGGS eukaryotic expression plasmid to obtain pCAGGS-Signal peptide-RBD-(Thrombin site)-Fc. The plasmid was purified from E. coli (DH5α) with an endotoxin-free plasmid extraction kit (Invitrogen, CA, USA) and transfected into ExpiCHO-S cells using an ExpiFectamine Transfection Kit (Gibco, NY, USA). Cell supernatants containing RBD-Fc were collected 8 days later and filtered with a 0.22-μm membrane before being subjected to affinity chromatography through Protein A agarose. The eluted fraction that contained RBD-Fc was collected for further analysis. The RBD without an Fc tag was obtained from RBD-Fc by removing the Fc region by thrombin digestion. The RBD proteins were collected from the flow-through faction, and affinity chromatography against Protein A was repeatedly conducted to completely remove the residual Fc regions.