Hitrap q column

We previously found that the active site of WT was occupied by Ni²⁺ ions taken up by nickel‐nitrilotriacetic acid affinity chromatography (Ni‐NTA) during purification [10 (link)]. Therefore, to investigate metal dependency, we avoided the use of Ni‐NTA to purify WT and TTE^Δ100.
Proteins from sonicated and heat‐treated solutions of recombinant E. coli were separated by AEX on a HiTrap Q column (Cytiva). The fusion tag was cleaved using PreScission protease (Takara Bio). Separation of the WT and the fusion tag was performed by AEX using a HiTrap Q column. The final purification step was size‐exclusion chromatography (SEC) using a HiLoad 26/600 Superdex 75 prep‐grade column (Cytiva).
Proteins from sonicated and heat‐treated solutions of recombinant E. coli were precipitated by a 40% saturation concentration of the ammonium sulfate fraction. The next purification step was AEX, using a HiTrap Q column. The separation of TTE^Δ100 and the fusion tag was performed by HIC using a HiTrap Butyl HP column. The final purification step was SEC using a HiLoad 26/600 Superdex 75 prep‐grade column. Purified WT and TTE^Δ100 were dialyzed overnight in the presence of 50 mm EDTA to remove metal ions, and the solvent was substituted with 50 mm sodium phosphate buffer (pH 8.0) containing 150 mm NaCl.

The scFv fragments fused with N-terminal N11-tag and TEV protease recognition sequences were expressed in an E. coli cell-free protein synthesis system as described above with the following exception: the synthesis mixture included 5 mM glutathione disulfide and 0.4 mg/mL disulfide-bond isomerase (DsbC) without reducing agent at 25°C. The synthesized proteins were purified by using the HisTrap column and then digested with TEV protease (47 (link)) and dialyzed against the same buffer including 20 mM imidazole, followed by a second HisTrap chromatography to remove cleaved His-tag and the His-tagged TEV protease. The desalted protein solution in 20 mM Tris-HCl buffer (pH 7.0) containing 80 mM NaCl was subjected to a HiTrap SP column (Cytiva) and eluted with a 0–1.0 M NaCl gradient. Eluted proteins were concentrated to 7 mg/mL.
The Fv-clasp fragments (27 (link)) derived from the CV-1A1 scFv with N-terminal N11-tag and TEV protease recognition sequences were prepared in the same manner as described above, except the synthesis mixture included 5 mM glutathione disulfide, 0.8 mg/mL DsbC, and 0.2 mg/mL Skp chaperone protein without reducing agent, and a HiTrap Q column (Cytiva) was used instead of a HiTrap SP column.

The Chiba P domain protein was expressed as N11-tagged SUMO fusions in an in vitro E. coli cell-free protein synthesis system (48 (link)). The fusion protein was immobilized by a HisTrap column (Cytiva, Piscataway, NJ), washed with 20 mM imidazole, and then eluted with 500 mM imidazole in 20 mM Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl. Fusion proteins were digested by SUMO protease while dialyzing against the same buffer including 20 mM imidazole, followed by a second HisTrap chromatography. The resultant flowthrough fraction containing cleaved P domain proteins was desalted by using a HiPrep Desalting column (Cytiva) [in 20 mM Tris-HCl buffer (pH 8.0) containing 10 mM NaCl] and then subjected to anion exchange chromatography using a HiTrapQ column (Cytiva). The P domain protein was eluted by applying a NaCl gradient. The peak fraction was concentrated by using an Amicon Ultra 15 centrifugal device (MWCO 10,000) (Merck Millipore, Burlington, MA) to a protein concentration of approximately 1 mg/mL in 20 mM Tris-HCl (pH 8.0).

NAD, NMN, Digitonin, Poly-L-lysine, KH₂PO₄, NH₄HCO₃, chloroacetamide, polyethylenimine (PEI), and urea were purchased from Sigma-Aldrich. DMEM, Trypsin-EDTA, penicillin/streptomycin solution, Lipofectamine 2000, formic acid, and acetonitrile were purchased from Thermo Fisher. FBS was obtained from PAN Biotech. SMM-293TII media was obtained from Sino Biological. Ni-Excel column, HiTrap Q column, CM5 sensor were obtained from Cytiva. Strep-Tactin resin, StrepTactin^TM XT SPR kit were obtained from IBA. General chemicals were purchased from Aladdin, Macklin, or Sangon Biotech (Shanghai).

Cells collected after induction were let thaw on ice and resuspended in 50 mL/L culture binding buffer (50 mM Na-phosphate, 300 mM NaCl, 10 mM imidazole, pH 8.0). The cell suspension was subjected to 4 cycles of sonication/rest of 2 min each at 80% power, keeping the solution on ice all the time. After cell disruption, cellular debris were removed by centrifugation followed by filtration using a 0.22 μm filter. Cleared cell extract was applied to a 5 mL HisTrap column (Cytiva Europe, Milan, Italy) equilibrated in binding buffer. Protein elution was obtained by an imidazole step gradient from 0 mM to 500 mM. Eluted proteins were checked by SDS-PAGE electrophoresis in reducing conditions. Fractions positive for sdASNase were pooled and applied to a HiTrap Desalting column (Cytiva Europe, Milan, Italy) equilibrated in 50 mM Na-phosphate pH 7.4. The obtained fractions were loaded onto a 1 mL HiTrap Q column (Cytiva Europe, Milan, Italy) equilibrated in 50 mM Na-phosphate pH 7.4. Protein elution was obtained by a linear NaCl gradient and the fractions obtained, along with the flow-through, were analyzed by electrophoresis in reducing conditions, as previously described [24 (link)].

Purified REV7 mutants at 500 µg in total were subjected to anion-exchange (AE) chromatography using a 1 ml Hi-Trap Q column (Cytiva). Protein samples were resuspended in low salt buffer B prior loading onto Q column. Bound proteins were eluted using a 20–500 mM NaCl gradient over 20 column volumes. Fractions collected from the run were analyzed by 12% SDS-PAGE.

We modified the purification to more reliably obtain FUS or mutant FUS LCR_N without the SUMO fusion tag. Nickel affinity chromatography and size exclusion chromatography were followed by consecutive purification on a HiTrap Q column (Cytiva) and an 8 ml phenyl Superose column (Cytiva) using buffer with 40 mM arginine, pH 9 and gradients from 50 mM to 1M NaCl and 1 M to 0 M NaCl, respectively. Following cleavage with ULP1 the protein was again purified by phenyl Superose using the same gradient, to yield highly pure FUS LCR_N.