Purification of A3A-MycHis for Deaminase Assays

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).