Histrap hp affinity column

The heavy and light chain plasmids of each antibody were transiently co-transfected into HEK293T cells at a ratio of 2:3 using polyethylenimine. After 6 h, the supernatant was replaced with DMEM without FBS. The supernatant was collected 3 days post-transfection for SPR analysis. The heavy and light chain plasmids of each antibody were also transiently co-transfected into HEK293F cells to express antibodies for the pseudovirus assays. Five days later, the HEK293F supernatants were collected, and antibodies were purified using 5-mL Protein A affinity columns (GE Healthcare). RBD proteins were expressed in HEK293F cells and purified using 5-mL HisTrap HP affinity columns (GE Healthcare). The soluble proteins were further purified by gel filtration using a Superdex™ 200 10/300 GL column (GE Healthcare). Fabs were generated by papain digestion and further purified using a Protein A column (S309 Fab and BD-604 Fab) or Resource Q column (S304 Fab) and gel filtration using a Superdex™ 200 10/300 GL column. RBDs and Fabs for cryo-EM were stored in buffer containing 20 mM Tris-HCl and 150 mM NaCl (pH 8.0). Antibodies and RBDs for SPR analysis were stored in PBS.

Soluble proteins were collected after centrifugation at 18,000 rpm for 30 min at 4°C and filtered. Subsequently, protein purification was carried out with a HisTrap HP affinity column (GE Healthcare Life Sciences) equilibrated with cell re-suspension buffer on an ÄKTA pure protein purification system (GE Healthcare Life Sciences). Elution of proteins was carried out in a gradient of equilibration buffer containing 20 mM Tris buffer (pH 8.0), 300 mM NaCl, 2 mM DTT and 1 M imidazole. Fractions containing EC12 were subjected to western blot analysis using anti-6xHis antibody. The fractions were pooled and separated on a HiTrap Q HP column (GE Healthcare Life Sciences) equilibrated with buffer containing 20 mM Tris (pH 8.0), 2 mM DTT and eluted in a gradient from 0 to 1 M NaCl. At the last step, the 6xHis tag on the recombinant protein was removed by a protease encoded by the TEV. HisTrap chromatography was then used to separate untagged recombinant proteins. The purified protein was concentrated and frozen for future use. EC12 mutants used in the competitive binding assay were obtained through the first HisTrap affinity purification without TEV cleavage.

E. coli JM109 and E. coli BL21 (DE3) were used as the hosts for recombinant gene cloning and expression, respectively. The recombinant plasmid pET28a-Btpul (GenBank Accession No. EMBL AJ315595) was constructed by Genewiz (Suzhou, China). The plasmid pEVOL-pBpF encoding AARS/tRNA was purchased from Addgene (Beijing, China). PrimeSTAR HS DNA polymerase, which was used for polymerase chain reaction (PCR), was purchased from TaKaRa Biotechnology Co., Ltd. (Beijing, China). Primers were all synthesized by Genewiz (Suzhou, China). The ClonExpress II One Step Cloning Kit for homologous recombination was purchased from the Vazyme Biotech Company, Ltd. (Nanjing, China). DNA sequencing was completed by Genewiz (Suzhou, China). The HisTrap HP affinity column and disposable PD-10 desalting column that were respectively used for protein purification and desalination were purchased from GE Healthcare Life Sciences (Beijing, China). O2beY, pullulan, and l-arabinose were purchased from Tokyo AMATEK (Suzhou, China), Chemical Industry (Tokyo, Japan), and Ourchem (Shanghai, China), respectively. Premixed Protein Marker (High) and 4× Protein SDS-PAGE Loading Buffer were purchased from TaKaRa Biotechnology Co., Ltd. (Beijing, China). All other materials were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Sinopharm Chemical Reagent (Shanghai, China).