Nickel nta

Recombinant GST-KHK-A, GST-YWHAH, and free GST proteins were expressed in Escherichia coli BL21 cells, pulled down using glutathione-affinity beads (GE Healthcare; Chicago, IL) at 4 °C for 1 h, and eluted with 10 mM reduced glutathione (Sigma-Aldrich). FLAG-LRRC59 and His(6)-KHK-A proteins were expressed in HEK293T cells, pulled down using EZview Red anti-FLAG (Sigma-Aldrich) and nickel-NTA (Qiagen, Hilden, Germany) affinity beads at 4 °C for 4 h, and eluted with 500 ng/μL of FLAG peptide and 250 mM imidazole, respectively. The amounts and purities of proteins were checked by SDS-PAGE and Coomassie Brilliant Blue R-250 staining.

The SiREM6 and StREM1 genes were cloned into the pET-28a vector containing a His tag. The recombinant vectors were independently transformed into Escherichia coli BL21 cells and then the cells were induced by 1 mM isopropyl-β-D-thiogalactoside (IPTG) for 4 h at 28°C. The fusion proteins were purified by nickel NTA (Qiagen, Germany).
For the negative-staining assay, recombinant remorins were dialyzed against 10 mM Tris (pH 7.5). The final protein concentrations were 80 µg/ml. Then, the recombinant remorins were adsorbed on formvar-coated copper grids for 10 min, stained with 2% uranyl acetate for 4 min, and air-dried. The samples were visualized at a magnification of 80000× using a Hitachi 7500 electron microscope (Japan). Photographs were taken using iTEM (OSIS, Germany).

The plasmid pET28b(+)-CRD-BP which contains the mouse CRD-BP cDNA was used to generate recombinant WT CRD-BP. The generation of various KH point mutation variants was accomplished using PCR-based site-directed mutagenesis method and has been previously described [21 (link)]. Recombinant CRD-BP was purified from Escherichia coli BL21 (DE3) using a 1 mL bed volume of nickel-NTA (QIAGEN) column under denaturing conditions. Proteins eluted from the column at pH 5.4 were subjected to three steps of dialysis. The first step was for 24 hours in pH 7.4 buffer containing 200 mM NaCl, 20 mM Tris-HCl, 1 mM reduced glutathione, 0.1 mM oxidized glutathione, 10% (v/v) glycerol, 2 M urea, and 0.01% (v/v) Triton X-100. Proteins were then dialyzed twice, each for 2 hours in the same buffer as above, but without urea and the glutathiones. Following dialysis, samples were spun at 13,200 rpm for 30 min to remove any precipitated proteins. The purified protein solutions were then quantified using Quick Start Bradford 1 x Dye Reagent (Bio-Rad, Mississauga, Ontario) or BCA Protein Assay Kit (ThermoFisher, Ottawa, Ontario), and analyzed for purity using Coomassie brilliant blue-stained 12% SDS-PAGE. The purified proteins were also confirmed by Western analysis using specific antibody against CRD-BP [21 (link)].

Proteins were prepared as previously described (13 (link)). Briefly, Pol γA WT, Exo-deficient [D198A/E200A (exo⁻)], and mutants [Pol γA R853A, Pol γA R853Q, Pol γA R853W, and Pol γA R853A (exo⁻)] were expressed in insect cells Sf9 and purified using TALON (Clontech) and Superdex 200 (Cytiva) column chromatography. Pol γB WT and a deletion mutant Pol γB-ΔI4 (deletion of amino acids 136 to 182) were expressed in E. coli Rosetta BL21 DE3 and purified using Nickel-NTA (Qiagen) and cation-exchange (Mono S) chromatography. For structural studies, purified Pol γA and Pol γB were mixed at a 1:2 molar ratio on ice for 10 min and purified using Superdex 200 (Cytiva) column chromatography. The protein purity was estimated to be >95% per SDS–polyacrylamide gel electrophoresis analysis.

Purification of A3A-MycHis as a positive control for deaminase activity assays was performed similar to as previously described.⁶² (link) First, 293T cells were transfected with pcDNA3.1-A3A-Myc-His. 48 h later, 1 × 10⁸ cells were harvested, washed with PBS, and resuspended in 10 mL of cell lysis buffer (25 mM HEPES, pH7.4, 300 mM NaCl, 20 mM imidazole, 0.1% triton X-100, 10 mM MgCl₂, 0.5% TCEP, and 10% glycerol). The cell suspension was sonicated using a Branson Sonifier 450 for 2 min at 40% duty cycle power 5. RNase A (Qiagen) and Benzonase were added to the suspension to 100 μg/mL and 5 μL/25 mL, respectively. These were then incubated at 37°C for an hour with nucleases and subsequently clarified by spinning at 16,000 g for 30 min at room temperature. NaCl was then added to these lysates to a final concentration of 1M. 50 μL of Nickel-NTA (Qiagen) superflow beads were added and mixed by rotating for 2 h at room temperature. This was then loaded onto a Poly-Prep chromatography column (Bio-Rad) and washed using wash buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 0.1% triton X-100, 40 mM imidazole, and 20% glycerol). The final protein was eluted using elution buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 0.1% triton X-100, 300 mM imidazole, 20% glycerol, and 1 mM TCEP) and concentration was determined using Bradford assays (Bio-Rad).