Vivaspin 500 mwco 50 000 device

Affinity chromatography for protein purification was performed as previously described^{33 (link)}. Harvested DH5α cells suspended in 40 mL PBST (1.37 M NaCl, 27 mM KCl, 81 mM Na₂HPO₄·12H₂O, 14.7 mM KH₂PO₄, and 0.05% Tween 20) were disrupted by sonication (VC-750, EYELA, Tokyo, Japan) 10 times for 20 s at 20% intensity. The disrupted cells were removed by centrifugation at 5800 × g for 2 min at 4 °C. The supernatant was transferred to a 50-mL tube and placed on ice, and 560 μL of Glutathione Sepharose 4B resin (GE Healthcare Japan) was mixed into the supernatant, followed by gentle shaking for 30 min. After centrifugation (5800 × g for 2 min at 4 °C), the supernatant was removed and the resin was re-suspended in 700 μL of PBST. After washing five times, recombinant proteins were eluted five times with 700 μL of GST elution buffer [50 mM Tris-HCl (pH 8.0), 10 mM reduced glutathione]. Proteins were concentrated with a Vivaspin 500 MWCO 50000 device (Sartorius, Göttingen, Germany), and protein concentrations were analysed with a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). SDS-PAGE was performed to analyse protein purification with staining using InstantBlue (Expedion Protein Solutions, San Diego, CA).

Centrifuged E. coli BL21(DE3) cells resuspended in 40 mL PBST (0.137 M NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄·12H₂O, 1.47 mM KH₂PO₄, and 0.001% Tween 20) were disrupted by sonication (VC-750, EYELA, Tokyo, Japan) 10 times for 20 s at 20% intensity. The insoluble fraction was removed from the disrupted crude extract by centrifugation at 5800 × g for 2 min at 4 °C. The supernatant was transferred to a 50 mL tube and washed with 700 μL of Glutathione Sepharose 4B resin (Global Life Sciences Technologies Japan K.K., Tokyo, Japan), and the mixture was gently shaken for 30 min. After centrifugation, the supernatant was removed, and the resin was resuspended in 900 μL of ice-cold PBST. After washing 10 times, the recombinant proteins were eluted five times with 700 μL of GST elution buffer (50 mM Tris-HCl [pH 8.0], 10 mM reduced glutathione). Proteins were concentrated using a Vivaspin 500 MWCO 50,000 device (Sartorius, Germany) and protein concentrations were analyzed using a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). SDS-PAGE was performed to evaluate protein purification by staining with Quick Blue Staining Solution (BioDynamics Laboratory Inc., Tokyo, Japan).

The recombinant E. coli DH5α cells from 800 mL culture were suspended in 40 mL of phosphate-buffered saline/tween (PBST) (1.37 M NaCl, 27 mM KCl, 81 mM Na₂HPO₄·12H₂O, 14.7 mM KH₂PO₄, and 0.05% Tween 20) and lysed through sonication (model VC-750; EYELA, Tokyo, Japan). The procedure was repeated 10 times for 10 s at 20% intensity. The lysed cells were centrifuged at 13,000 × g for 15 min at 4 °C. The supernatant was transferred to a 50-mL tube, and 560 µL of Glutathione Sepharose 4 B resin (GE Healthcare Japan, Tokyo, Japan) was added. Thereafter, the mixture was gently shaken for 30 min on ice. To remove the supernatant, the mixture was centrifuged at 5,800 × g for 2 min at 4 °C. The resin was re-suspended in 700 µL of PBST and washed five times. After washing, the recombinant protein was eluted with 700 µL of glutathione-S-transferase (GST) elution buffer (50 mM Tris–HCl (pH 9.6) and 10 mM reduced glutathione) five times, and the protein was concentrated using a Vivaspin 500 MWCO 50,000 device (Sartorius, Göttingen, Germany). The protein concentration was measured using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). To verify protein purification, sodium dodecyl sulphate–polyacrylamide gel electrophoresis was carried out, and the gel was stained using Instant Blue reagent (Expedeon Protein Solutions, San Diego, CA, USA).