Ni affinity chromatography

A 336 bp cDNA fragment encoding the entire Lj-BLNK SH2 domain (GenBank accession number: KF692036) was cloned into a pMD19-T vector (TaKaRa Biotechnology, Dalian, China) as described in the Supplementary Information. This truncated recombinant Lj-BLNK fragment (rLj-BLNK) flanked by XhoI and EcoRI restriction sites was cloned into the expression vector pET-28a (+) with a His-tag. rLj-BLNK was expressed in E. coli BL21with 0.25 mM isopropyl β-D-thiogalactopyranoside (IPTG) (TaKaRa Biotechnology, Dalian, China) induction for 3.5 h. Subsequently, the bacterial cells were harvested by centrifugation at 12000 rpm for 15 min at 4 °C. The cell pellet was suspended in Tris-HClbuffer (50 mM, pH 7.0) containing 20 mM NaCl and 10 mM imidazole, then lysed by sonication. The soluble supernatant was collected and purified with Ni affinity chromatography (GEHealthcare, New York, NY, USA). The concentration of rLj-BLNK was measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Purified rLj-BLNK was analyzed by 15% SDS-PAGE and stored at −20 °C.

The PDCoV M^pro coding sequence was cloned into the BamHI and XhoI restriction sites of the pET-28b_SUMO vector and then transformed into Escherichia coli strain BL21 (DE3). The fusion protein SUMO-PDCoV M^pro was purified by Ni-affinity chromatography (GE Healthcare, Uppsala, Sweden) and then cleaved with ULP protease. M^pro was further purified using anion exchange chromatography (HiTrap Q, GE Healthcare, Uppsala, Sweden) with a linear gradient from 2.5 to 500 mM NaCl (20 mM Tris-HCl pH 8.0) and size exclusion chromatography (Superdex 75 10/300 GL, GE Healthcare, Uppsala, Sweden) in 10 mM HEPES pH 7.5 and 150 mM NaCl.

Soluble scFvs were expressed in E. coli Mach1 cultured at 37 °C in 2×YT medium supplemented with 0.1% glucose and 100 μg/mL ampicillin. When bacterial growth reached 0.8 OD600, the expression was induced with 1 mM IPTG. E. coli was further propagated at 30 °C for 10 h, followed by centrifugation at 4000 g. A fully-automated liquid chromatography instrument Äkta (GE Healthcare) was used for affinity chromatography purification. ScFv-enriched supernatant was filtered and purified with immobilized metal affinity chromatography (IMAC) using protein A (GE) or with Ni-affinity chromatography (GE). Buffers used were as follows. For protein A chromatography purification: binding buffer, 20 mM Tris pH 8.0; washing buffer, 20 mM glycine pH 6.0; and elution buffer, 0.1 M glycine pH 2.5. For Ni-affinity chromatography purification: binding buffer, 50 mM sodium phosphate, 300 mM NaCl, 10 mM imidazole, pH 7.4; and elution buffer, 50 mM sodium phosphate, 300 mM NaCl, 500 mM imidazole, pH 7.4. Eluted fractions (2 mL) containing scFvs were pooled together, concentrated up to 1 mg/mL, and dialyzed overnight against PBS, pH 7.4, at 4 °C. NanoDrop A280 measurements were used for estimating soluble protein concentrations, and the obtained yield of scFvs was in the range of 1–4 mg from 1 L of bacterial culture.

Recombinant Tcnα, TcdB, TpeL, TcsL, and TcsH were expressed in Bacillus subtilis SL401 and purified as His-tagged proteins^{52 (link)}. In brief, B. subtilis cells were cultured at 37 °C till OD₆₀₀ reached 0.6 and then induced with 1 mM isopropyl-β-D-thiogalactoside at 25 °C for 20 h. The recombinant LDLR_LA-Fc with His-tag at C-terminus was expressed in the Expi293F cells. In brief, 5 × 10⁸ Expi293F cells were transfected with 750 μg of pHLsec-LDLR_LA-Fc using 1 mg/ml PEI. The supernatant was collected 4 days post-transfection and applied to purification. All above recombinant proteins were purified by Ni-affinity chromatography and size-exclusion chromatography (GE Healthcare).

The previously constructed recombinant plasmids were transformed into E. coli BL21(DE3) competent cells (TransGen) by heat shock at 42°C for 30 s and screened with kanamycin (50 μg/ml). Single colonies were selected from the plate and inoculated into LB medium with kanamycin (50 μg/ml) at 37°C. When the OD600 was 0.6, isopropyl-b-D-thiogalactoside (0.2 mM) was added to the culture medium and further incubated for 6 h at 37°C. The cells were centrifuged for 20 min at 8,000 ×g to collect the bacteria. Sonication was performed, and the supernatant was separated and precipitated by centrifugation for 20 min at 8,000 ×g. The precipitate was resuspended in Tris-HCl (20 mmol/l, pH 8.0) buffer containing 8 M urea at a ratio of 1:10 (w/v). The mixture was incubated at 4°C overnight and then filtered through a 0.45 μm membrane. The denatured p170 and p170-RBD proteins were then further purified with Ni-affinity chromatography (GE Healthcare, USA) using the ÄKTA explore system (GE Healthcare). The column was equilibrated with Tris-HCl buffer (20 mmol/l, pH 8.0) containing 8 M urea. The concentration of imidazole was increased gradually, and the corresponding peak was collected. Purified protein was refolded by reducing the urea concentration gradually with a dialysis bag, followed by concentration with 10 kDa and 100 kDa ultrafiltration devices successively and then stored at -80°C.

Expression of Cry2Ah1 protein and Cry2Ah1-vp and Cry2Ah1-sp in 300 mL LB medium of E. coli was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16 °C overnight, and then cells were collected by centrifugation at 10,000 g for 8 min and the cell pellet was resuspended in 30 mL of binding buffer (20 mM Tris-HCl with 0.5 M NaCl and 50 mM imidazole, pH 8.0). Cells were then lysed by ultrasonication (Ningbo Scientz Biotechnology Co., Ltd., Ningbo, China) for 6 min (70% power, 3-s pulse on, 5-s pulse off) and then centrifuged at 13,000 g for 15 min at 4 °C. Cry2Ah1 toxin and Cry2Ah1-vp and Cry2Ah1-sp were solubilized from inclusion bodies with 5 mL urea buffer (8 M urea, 10 mM Tris-HCl and 100 mM NaH₂PO₄). These three proteins were purified by Ni-affinity chromatography (GE Healthcare, Uppsala, Sweden), then preequilibrated with urea buffer. Nonspecifically adsorbed proteins were removed by washing with decreased concentration urea buffer (6 M urea, 4 M urea and 2 M urea) orderly. Proteins bound to the column were eluted with elution buffer (20 mM Tris-HCl, 500 mM NaCl, 250 mM imidazole, pH 8.0).