Histrap high performance column

The SARS-CoV-2 HexaPro S glycoprotein ectodomain construct comprises residues 1–1208 with the native signal peptide, the HexaPro prefusion stabilizing mutations (F817P, A892P, A899P, A942P, K986P, V987P), abrogation of the furin cleavage site (residues 682–685, GSAS) and a C-terminal short linker (GSG), followed by a foldon, HRV 3C site (LEVLFQGP), a short linker (GSG), an avi tag, a short linker (GSG), an 8x his tag in a pcDNA3.1(−) plasmid. The SARS-CoV-1 HexaPro S glycoprotein was expressed as previously described. Expi293F cells were grown at 37°C with 8% CO₂ and DNA transfections were conducted with the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were harvested four days post-transfection and proteins were purified using HisTrap^™ High Performance column (Cytiva). Proteins were first washed with 10–15 column volumes of a buffer containing 25 mM sodium phosphate, 300 mM NaCl, 20 mM imidazole, pH 8.0, followed by elution with 10–15 column volumes using 300 mM imidazole, pH 8.0. Eluted proteins were concentrated and buffer exchanged into 1x TBS (20 mM Tris, 150 mM NaCl, pH 8.0) using Amicon Ultra-15 Centrifugal Filter Unit (100 kDa) (Millipore). Purified proteins were snap frozen and stored at −80°C.

Pilot release kinetics were performed using 5,5,5-trifluoro-DL-leucine (Synquest Laboratories; 3x activated off ribosome). CCA trinucleotide was synthesized by Dharmacon, Inc. Tritium-labeled Met was purchased from PerkinElmer. All other chemicals and reagents were purchased from Sigma–Aldrich or Merck. Escherichia coli–methylated RF1 with a C-terminal His₆ tag was co-overexpressed with untagged HemK methyltransferase and purified by a HisTrap High-Performance column (Cytiva) as previously described (13 ). Other translation factors, E. coli MRE600 70S ribosomes, and f[³H]Met-tRNA^fMet from overexpressed tRNA were prepared as described previously (16 (link)) (and references therein). All experiments were done at 37 °C in a Hepes–polymix (PM) buffer containing 5 mM Mg(OAc)₂, 0.5 mM CaCl₂, 95 mM KCl, 3 mM NH₄Cl, 1 mM spermidine, 8 mM putrescine, 1 mM dithioerythritol, and 30 mM Hepes, in the absence or the presence of organic solvents. The pH of the PM buffer was adjusted with 0.5 M KOH or 1 M HCl.

The PDCoV_{IL121_2014} RBD (Genbank KJ481931.1) and SD2018/300 (Genbank KJ481931.1) spanning residues 303–415 were cloned into pcDNA3.1+ plasmid by GenScript with an N-terminal mu-phosphatase signal peptide sequence and C-terminal short linker GSG, AVI tag and 8 x His tag. The DNA constructs were expanded using DH5ɑ cells and purified using Qiagen MegaPrep Kit. Protein was expressed using ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Expi293F cells were grown at 37°C with 8% CO₂ to 3E6 cells and transfected with 100 μg of DNA. Cell culture supernatants were harvested four days post-transfection. Protein was purified using HisTrap^Tm High Performance column (Cytiva). Protein was bound to HisTrap resin in 50mM Tris-HCl, 150mM NaCl, 10mM Imidazole at pH 8.0. Unbound proteins were washed in 10 column volumes of 50mM Tris-HCl, 150mM NaCl, 10mM Imidazole at pH 8.0. Eluted with 50mM Tris-HCl, 150mM NaCl, 250 mM Imidazole at pH 8.0. The eluted RBD was concentrated using Amicon Ultra-15 Centrifugal Filter Unit (10 kDa) and buffer exchanged into 50mM Tris, 150mM NaCl pH 8.0 and further purified using size exclusion chromatography Superdex 200 10/300. Endotoxin levels were assessed using Charles River Limulus Amebocyte Lysate (LAL) cartridges. Proteins were snap frozen in liquid nitrogen and stored at −80°C.