Hiload superdex 200 pg column

FreeStyle 293-F cells (Thermo Fisher) were grown in FreeStyle 293 expression medium (Thermo Fisher) according to the manufacturer’s recommendations. The temperature was 37 °C, the shaker speed was 130 rpm, and the CO₂ pressure was 8%. Transfection was carried out on freshly passaged cells that had been adjusted to a density of 2 × 10⁶ cells/mL. Expression plasmids for the heavy chain and the light chain were added to the culture at final concentrations of 1.5 µg/mL each. Polyethylenimine-25K (Polysciences) was added as a transfection agent at a final concentration of 9 µg/mL. Twenty hours after transfection, the culture was supplemented with 3 mg/mL glucose and 3.7 mM valproic acid (Sigma). Culture supernatants were harvested 5–6 days after transfection when cell viability dropped below 70%. Secreted antibodies were bound to a HiTrap 1 mL Protein A HP column (GE Healthcare) that was subsequently washed with 10 mL of 20 mM Tris-Cl, 150 mM NaCl. Antibodies were eluted in 50 mM phosphate/citrate buffer at pH 4.0 and were immediately pH-adjusted with 200 mM Tris-Cl pH 8. The antibodies were further purified by gel permeation chromatography on a HiLoad Superdex 200 pg column (GE Healthcare) in 20 mM Tris-Cl pH 8.0, 150 mM NaCl.

E. coli BL21 (DE3) pLysS cells transformed with pET23b-FB-GFP were grown at 37 °C to a density of OD600 0.6 in 2xYT medium containing Ampicillin (100 mg L⁻¹) and Chloramphenicol (25 mg L⁻¹) with shaking. Isopropyl-β-d-thiogalactoside (IPTG) was added to induce protein expression at a final concentration of 0.4 mM, and the temperature was lowered to 18 °C. Cells were harvested after 16 h by centrifugation and resuspended in PBS buffer supplemented with 300 mM NaCl, protease inhibitors (ThermoFisher), 0.2 mM AEBSF (20 mL L⁻¹ culture) and lysed by sonication. Lysate was clarified by centrifugation. The supernatant was loaded onto two connected HisTrap HP 5 mL columns (GE Healthcare), washed and eluted by a linear gradient of 0–500 mM imidazole. The fractions containing the POI were pooled, concentrated using Amicon Ultra-15 30 kDa MWCO centrifugal filter unit (EMD Millipore) and loaded onto a size-exclusion HiLoad Superdex 200 PG column (GE healthcare) in HEPES-based buffer (25 mM HEPES pH 7.9, 12.5 mM MgCl₂, 100 mM KCl, 0.1 mM EDTA, 0.01% NP40, 10% glycerol, and 1 mM DTT). The fractions containing FB-GFP protein were collected, concentrated, and stored at −80 °C after flash freezing by liquid nitrogen.

The complex of the SBV Gc head domain (aa 465–702) with scFv 1C11 was purified from an equimolar mixture by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl on a HiLoad Superdex 200 pg column (GE Healthcare). Optimal crystals were obtained by the hanging-drop vapor diffusion method: 1.0 µL of a 21.7 mg/mL solution of the complex in 20 mM Tris-Cl pH 8.0, 150 mM NaCl was added to 0.75 µL of reservoir solution containing 0.1 M sodium acetate, 8% v/v 2-propanol, 25% w/v PEG 4 K. The drops were equilibrated against reservoir solution on siliconized glass slides (Hampton) for 5 days at 18 °C. Crystals were cryo-protected by immersion in 25% v/v glycerol, 75 mM sodium acetate, 6% v/v 2-propanol, 19% w/v PEG 4 K for 10 s prior to conservation in liquid nitrogen.

The COMP sequence was obtained from UniProtKB with accession number P49747. The construct contains the native leader sequence and the full-length COMP sequence and the C-terminal his-tag. The gene was synthesized at Eurofins with KpnI and XhoI restriction sites at the 5’ and 3’ ends. The synthesized gene was restriction enzyme digested using FastDigestTM enzymes (ThermoFisher Scientific). The digested DNA fragment was cloned into a mammalian expression vector pCEP4 (Life technologies) that was digested using the same restriction enzymes. After sequence verification, the recombinant plasmid was transfected into Expi393FTM cells (Life technologies) using the FectoPROTM DNA transfection reagent (Polyplus transfection). The supernatants were harvested 6 days post transfection. The recombinant protein was first captured using a 5 ml HisTrap Excel (GE Healthcare Life Sciences) affinity column followed by size exclusion chromatography on a HiLoad Superdex 200 pg column (GE Healthcare life Sciences) equilibrated in PBS. The purified recombinant protein (Supplement Figure 1) was obtained as a single peak and was concentrated using an Amicon centrifuge device with MWCO of 30 kD. The protein concentration was determined using absorbance at 280 nm.

Initial attempts to produce two different constructs of the LACV Gc head domain were unsuccessful. However, the complete variable region (aa 477–911) could readily be produced in S2 cells. Following affinity purification, the protein was further purified by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl on a HiLoad Superdex 200 pg column (GE Healthcare). As no crystals could be obtained with this construct, a sample of 0.7 mg/mL was proteolytically trimmed with 2 µg/mL trypsin (Sigma-Aldrich) for 30 min at 24 °C. The reaction was stopped with 5 µg/mL soybean trypsin inhibitor (Sigma-Aldrich), and the largest cleavage product with an apparent molecular mass of ~30 kDa was again purified by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl. Optimal crystals of this fragment were obtained by the hanging-drop vapor diffusion method: 0.75 µL of a 11.1 mg/mL protein sample in 20 mM Tris-Cl pH 8.0, 150 mM NaCl were added to 0.75 µL of reservoir solution containing 0.1 M HEPES pH 7.5, 10% v/v 2-propanol, 20% w/v PEG 4 K. The drops were equilibrated against reservoir solution on siliconized glass slides (Hampton) for 5 days at 18 °C. Crystals were cryo-protected by immersion in 17% v/v glycerol, 83 mM HEPES pH 7.5, 8.3% v/v 2-propanol, 16.6% w/v PEG 4 K for less than 30 s prior to conservation in liquid nitrogen.

The methods used to express in Chinese hamster ovary (CHO) cells and purify Fc-fused bsAbs have been described previously^{51 (link)}. Briefly, the Fc-fused bsAbs were first purified on a protein A column (GE Healthcare Bio-Science Corp., Piscataway, NJ, USA). Gel filtration analysis with a HiLoad Superdex 200 pg column (26/600; GE Healthcare) was used to fractionate the monomers of each bsAb. The column was equilibrated with phosphate-buffered saline (PBS), and protein A-purified bsAb was loaded onto the column at a flow rate of 2–2.5 mL/min. The long-term stability of bsAbs in storage was evaluated using a Superdex 200 GL column (10/300; GE Healthcare). The column was equilibrated with PBS, and purified bsAb was loaded onto the column at a flow rate of 0.5 mL/min. For Western blotting analysis, hEx3-scDb-Fc(H237Y)-LH was prepared using Expi293 Expression System (Thermo Fisher Scientific, Waltham, MA, USA).

The complex of the SBV Gc head domain (aa 465–702) with scFv 4B6 was purified from an equimolar mixture by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl on a HiLoad Superdex 200 pg column (GE Healthcare). Optimal crystals were obtained by the hanging-drop vapor diffusion method: 0.75 µL of a 22.8 mg/mL solution of the complex in 20 mM Tris-Cl pH 8.0, 150 mM NaCl were added to 0.75 µL of reservoir solution containing 0.1 M sodium acetate pH 4.6, 3.5 M sodium formate. The drops were equilibrated against reservoir solution on siliconized glass slides (Hampton) for 5 days at 18 °C. Crystals were cryo-protected by immersion in 143 mM sodium acetate pH 4.6, 5 M sodium formate for less than 30 s prior to conservation in liquid nitrogen.