Sephacryl s 200 hr

Cloning of wild-type and mutant PfISN1 into pET-21bN was carried out using standard procedures as described in Supplementary Methods. For protein expression, either BL21 (DE3) or Rosetta strains were used. The cell pellet was re-suspended in 30 mL lysis buffer and lysed using French^© pressure cell press (Thermo IEC Inc., USA) over six cycles at 1000 psi. The components of the lysis buffer were 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% (w/v) glycerol, 0.1 mM PMSF and 0.5 mM Tris-(2-carboxyethyl) phosphine. The lysate was centrifuged at 14,000 × g for 45 min at 5 °C and the supernatant bound to Ni-NTA agarose beads (NI-NTA His-Bind^® Resin, Qiagen) for 3 h at 5 °C. Post binding, the beads were loaded onto a glass column and washed with at least ten equivalent of bead volume of lysis buffer containing increasing concentrations of 0, 20 and 40 mM imidazole. The protein was eluted in 5 mL of lysis buffer containing 500 mM imidazole. One millimolar of EDTA was added to chelate Ni²⁺ ions that could have eluted along with the protein. The eluted protein was concentrated using Amicon^® Ultra Centrifugal filter with a 30 kDa molecular weight cut-off (Millipore™ Corporation) and loaded onto a 16 mm × 60 cm column packed with Sephacryl™ S-200 HR (GE Healthcare Life Sciences).

After removing the dry outer membranaceous scales, bulb tissue samples were obtained from garlic and onion and stored in 4°C. Garlic (100 g) and onion (150 g) samples were used for GGT isolation by preparing a homogenate using phosphoric acid buffer (pre‐cooled, 50 mM NaH₂PO₄/Na₂HPO₄, pH7, 10% glycerol; 5% NaCl, 5 mM EDTA‐Na₂, 25 μM pyridoxal phosphate, 1 mM PMSF [benzyl sulfonyl fluoride], and 0.05% DTT [dithiothreitol]). The homogenate was spun at 10,000 g in a refrigerated centrifuge for 30 min, and the supernatant was separated using the methods of (NH₄)₂SO₄ precipitation and Gel filtration chromatography (Sephacryl S‐200 HR (2.5 × 42 cm², GE Healthcare), elution buffer, 50 mM NaH₂PO₄/Na2HPO4, pH7, 1.9 ml/min), as previously described (Shaw et al., 2005; Zhao & Qiao, 2009). Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) with 10% polyacrylamide gels and Coomassie brilliant blue (CBB) R‐250 staining was utilized to detect the GGT purity.

For expression of recombinant proteins, each pET17b vector containing the MvicOBP3 sequence was used for transformation into BL21(DE3) pLysS E. coli cells. The protein expression was induced at A₆₀₀ of 0.5–0.8 by addition of IPTG to a final concentration of 1 mM when the culture had reached a value of OD₆₀₀ = 0.8. The cells were grown for a further 3 h at 37 °C, then harvested by centrifugation and sonicated. After centrifugation, the OBP was present as inclusion bodies and solubilisation was performed by denaturation in urea/DTT, renaturation and extensive dialysis against 20 mM Tris buffer pH 7.4, using a method described previously19 (link). The protein was purified by anion-exchange chromatography as described previously47 (link) on XK 26 column (Pharmacia, Uppsala, Sweden) filled with DE-52 resin (Whatman, Kent, UK), followed by gel filtration on Sephacryl S-200 HR (GE healthcare, Piscataway, NJ). The purified protein was stored at –20 °C in 20 mM Tris–HCl at pH 7.4. The gel filtration buffer is 20 mM Tris plus 150 mM NaCl at pH 7.4, and the storage buffer is 20 mM Tris pH 7.4. The protein was desalted prior crystallisation by dialysis with 20 mM Tris pH 7.4 buffer.

The inhibition of LSD1 activity was evaluated according to reported references [23 (link),24 (link)]. pET-28b-LSD1 (full length) was transfected into BL21 (DE). Then, the protein was induced with 0.25 mmol/L IPTG following sonication and purified with Ni-NTA (Qiagen, Tubingen, Germany), Resource Q (GE, Pittsburgh, PA, USA) and Sephacryl S-200 HR (GE, Pittsburgh, PA, USA). The fluorescence intensity was read using EnVision Plate Reader (PerkinElmer, Waltham, MA, USA) to calculate the inhibition rate. The MAO-A and MAO-B were purchased from Active Motif (Cat#31502, Cat#31503, Carlsbad, CA, USA). Biochemical Kits were purchased from Promega (MAO-Glo Assay, Madison, WI, USA). The inhibitory activities of MAO-A and MAO-B were obtained according to the reported reference [25 (link)]. The inhibitory activities of CDK1 and CDK2 were obtained according to the reported reference [26 (link)].

Gel filtration was performed with ÄKTA start on a 60 cm × 16 mm column (cat. no. 19-5003-01, GE Healthcare, Chicago, IL, USA), packed with Sephacryl^® S-200 HR (cat. no. 17-0584-10, GE Healthcare). The column was equilibrated with 50 mM phosphate buffer, 150 mM NaCl, pH 7.4., and 0.25 mg plant-produced dRBD and gRBD proteins, purified using FLAG affinity chromatography, were loaded onto a column. Eluted fractions were combined and concentrated, and buffer exchanged against PBS and concentrated with a Millipore 10K MWCO Amicon Ultra 4 concentrator (cat. no.: UFC8010, Millipore).

pET-15 vectors for RRM2 (wild type and C244S) encoding residues 191–265 of human TDP-43 were purchased from GenScript. C198S plasmid was made in-house using a QuikChange II kit from Stratagene. The protein additionally containing N-terminal methionine was expressed in Rosetta DE3 cells (Novagen) using minimal M9 media (when necessary supplemented with ¹⁵N ammonium chloride and ¹³C glucose); expression was induced when OD600 reached 0.8. After 16 hours of incubation at 37 °C, cells were harvested, resuspended in the standard lysis buffer (containing 10 mM of β-mercaptoethanol) and then homogenized by SPEX SamplePrep 6870 Freezer/Mill followed by three cycles of sonication on ice. The sample was purified using anion-exchange and size-exclusion chromatography (columns GE HiTrap Q HP and GE Sephacryl S-200 HR). For various experimental measurements we used samples containing 1 mM RRM2, 20 mM sodium phosphate, 150 mM NaCl at pH 6.7, temperature 25 °C (standard sample conditions) unless indicated otherwise.

To remove all low molar mass components, the IEC-separated fractions were separated in a system consisting of a 15 × 360 mm precolumn packed with Toyopearl HW 40-S (Tosoh Bioscience GmbH), followed by 15 × 1070 mm Sephacryl S-200 HR and 15 × 1200 mm Sephacryl S-1000 SF columns (GE Healthcare Life Sciences, Uppsala, Sweden). The eluent was 50 mM AFB, pH 5.6 at a flow rate of 0.66 mL/min. Detection was achieved with a differential refractive index detector, and the system was calibrated using standard dextrans.
Ca. 120 mg of IEC fraction was dissolved in 1.5 mL of eluent and injected into the system. Salts and oligosaccharide containing fractions were discarded, and the lower molar mass peaks were separated from the high molar mass polysaccharide-containing fractions. Similar fractions were pooled together, frozen, and lyophilized twice in order to ensure the removal of the volatile salts.