Superdex g75 column

The bromo1 and bromo2 domains of BRD4 were expressed with an N-terminal HIS 10-SUMO tag and N-terminal GST-tag, respectively, in Escherichia coli strain BL21 (DE3) in the presence of 0.2 mM isopropyl β-d-thiogalactoside (IPTG) at 18 °C in LB medium. After overnight induction, cells were harvested by centrifugation and re-suspended in lysis buffer: 500 mM NaCl, 20 mM Tris-HCl, pH 7.5, and then disrupted by an EmulsiFlex-C3 high-pressure homogenizer (Avestin). Cell lysates were subjected to centrifugation at 16,770 × g, and the supernatants were applied to a GST affinity column or His affinity column. Proteins were directly digested on the column overnight by the PreScission proteases or SUMO protease ULP1. The resultant proteins were further purified by anion-exchange chromatography using HiTrap Q columns (GE Healthcare), followed by use of size exclusion chromatography Superdex G75 columns (GE Healthcare) with the elution buffer containing 20 mM Hepes-Na, pH 7.5, 100 mM NaCl, and 5 mM DTT. The peak fractions were pooled and concentrated to ~9 mg ml⁻¹, aliquoted and stored at −80 °C for future use.
Mutant BRD4-Bromo2 N433A, BRD2_bromos1/2, BRD3_bromos1/2, and Bromo1-linker(GGS)₅-Bromo2 of BRD4 were purified using the same procedure as described above.

Aβ1-40 (synthesized at Brigham and Women’s Hospital Biopolymer Laboratory) was dissolved in 6 M Guanidine HCl at 1 mg/ml, spun at 13,000× g for 10 min and purified by size-exclusion chromatography (SEC) isocratically using a Superdex G75 column (GE Healthcare, Chicago, IL, USA) and 70 mM NaCl + 5 mM Tris buffer, pH7.4. Aβ was eluted as a single peak and during purification was exchanged into the SEC buffer (i.e., removing the guanidine HCl). Experiments in western blot represents 11.3 μM purified Aβ alone or with 50 μM CuCl₂ and/or 250 μM H₂O₂ at 37 °C for 3 h. After incubation period samples were spun 3 times through a 3 kD MW cutoff filter using the SEC buffer as a wash buffer to remove Cu and H₂O₂. Western blot represents 20 μL of reaction mixture in which 1× loading buffer was added and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 16% Tris-tricine gel (Invitrogen, Carlsbad, CA, USA). Bands were visualized using anti-Aβ antibody (6E10, Signet Laboratories Inc., Dedham, MA, USA), HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and a chemiluminescent detection method per manufactures protocol (ECL Plus, GE Healthcare, Chicago, IL, USA).

Crotalus adamanteus venom (1 g) was dissolved completely in 10 ml of 1 M Tris-HCl (pH 7.4), and after centrifugation at 1800×g for 15 min, the clear yellowish solution was sterilized by filtration through a 0.22 µm membrane filter (Nalge Nunc International, Rochester, New York, USA). Venom samples (1 ml) diluted in 9 ml of Tris-HCl was then injected into a Superdex G-75 column attached to a high performance liquid chromatography system (HPLC, AKTA GE Healthcare, Denmark). Eluents (1 ml fractions) were monitored at 280 nm and collected in 1.5 ml eppendorf tubes. All the fractions were tested for enzymatic and antibacterial effects, and the fraction that showed the highest enzymatic and antibacterial effects were then subjected to reverse-phased liquid chromatography (RP-HPLC) via a C18 column (4.6×150 mm, Phenomenex, Upsala, Sweden), using a linear gradient of aqueous acetonitrile (80%) in 0.1% trifluoroacetic acid (Sigma Aldrich Co, St Louis, Missouri, USA) at a flow rate of 1.0 ml per min. Subsequent purification of active fractions were derived by using a C8 RP-HPLC column (100 Å, 4.6×250 mm) [1] .

The Tudor domain of Arabidopsis thaliana RDM15 (residues 598–662) was cloned into a pET-Sumo vector to fuse a hexahistidine and yeast Sumo tag to the target protein. The recombinant protein was expressed in E. coli strain BL21(DE3) by IPTG induction with a concentration of 0.3 mM. The protein was purified using a HisTrap (GE Healthcare) column. The His-Sumo tag was removed by ulp1 digestion followed by a second step with the HisTrap column. The target protein was further purified using a Superdex G75 column (GE Healthcare). The Se-Met labeled protein was expressed in Se-Met (Anatrace) containing M9 medium and was purified using the same protocol as used for the WT protein. All of the mutants were generated using a PCR-based mutagenesis method and were expressed and purified using the same protocol as used for the WT protein. All peptides used in this research were synthesized by GL Biochem (Shanghai).

HiPIP was extracted from cells of T. tepidum and purified as reported previously [14 (link),17 (link)]. Oxidation of HiPIP was done by adding of 10 mM K₃[Fe(CN)₆] just before use, because the purified sample was in the reduced state. The reagent was removed by size exclusion chromatography with a Superdex G-75 column (GE Healthcare). The sample in the reduced state was prepared by adding 10 mM dithiothreitol (DTT) in order to suppress by oxidation with O₂ during storage for a few days at 277 K. DTT was removed by size exclusion chromatography in the same way just before use.

The genes encoding SBPs from R. pomeroyi DSS-3 or R. nubinhibens ISM were amplified from the genome of R. pomeroyi DSS-3 or R. nubinhibens ISM by PCR using FastPfu DNA polymerase (TransGen Biotech, China). The dmpX homologue from P. sp. strain HTCC7211 (locus tag HTCC7211_00013840) was synthesized by the Beijing Genomics Institute (China). The related genes were then cloned into the NdeI/XhoI restriction sites of the pET-22b vector (Novagen, Germany) with a C-terminal His tag. Point mutations in DmpX were introduced using the PCR-based method and verified by DNA sequencing. The SBPs and corresponding mutant forms were produced in E. coli BL21 (DE3). The cells were cultured in the LB medium with 0.1 mg/ml ampicillin at 37 °C to an OD₆₀₀ of 0.8–1.0 and then induced at 18 °C for 16 h with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). After induction, cells were collected by centrifugation, resuspended in the lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 0.5% glycerol, pH 8.0), and lysed by a pressure crusher. The proteins were first purified by affinity chromatography on a Ni²⁺-NTA column (GE Healthcare, US), and then fractionated by gel filtration on a Superdex G75 column (GE healthcare, US).

The human Bcl-X_L lacking the C-terminus domain (Δ209-233) was expressed as a 6His-tagged protein in Escherichia coli strain BL21(DE3). Transformed bacteria were grown at 37 °C, 220 rpm up to an Optical Density (OD_600 nm) of 0.6–0.8, and protein expression induced overnight at 20 °C, 220 rpm with 1 mM IPTG. The unlabelled protein was expressed in LB medium while 15 N-labelled protein was produced in M9 minimal medium supplemented with 1 g/L 15NH4Cl and, 4 g/L unlabelled glucose. Ampicillin (100 µg/mL) was added as a selection agent. Cells were harvested by centrifugation (5000 g, 20 min, 4 °C) and resuspended in 20 mM PO4 (pH 8.0), 500 mM NaCl, 15 mM imidazole, and 10 mM β-mercaptoethanol, supplemented by Roche cOmplete protease inhibitors cocktail. The 6xHis-tagged Bcl-X_L was purified by Ni²⁺-affinity chromatography on a HisTrap FF crude column (GE Healthcare). To ensure the high final purity of Bcl-X_L, a further step of purification was carried out by size exclusion chromatography (SEC) on a Superdex G-75 column (GE Healthcare), equilibrated within 20 mM TRIS (pH 7.4), 150 mM NaCl and 1 mM DTT.