Escherichia coli BL21 DE3 Star (Stratagene) cells were transformed with the pColdII His-6-SUMO-APOBEC3A(E72A) vector described above. The E72A mutation was chosen to render the protein inactive. Expression occurred at 16 °C for 22 hours in lysogeny broth medium containing 0.5 mM IPTG and 100 µg/mL ampicillin. Cells were pelleted, re-suspended in purification buffer (50 mM Tris-HCl [pH 7.4], 300 mM NaCl, 1 mM DTT) and lysed with a cell disruptor. Cellular debris was separated by centrifugation (45,000 g, 30 min, 4 °C). The fusion protein was separated using HisPur Ni-NTA resin (Thermo Scientific). The His-6-SUMO tag was removed by means of a Ulp1 protease digest overnight at 4 °C. Untagged A3A(E72A) was separated from tag and Ulp1 protease using HisPur Ni-NTA resin. Size-exclusion chromatography using a HiLoad 16/60 Superdex 75 column (GE Healthcare) was used as a final purification step. Purified recombinant A3A was determined to be free of nucleic acid prior to binding experiments by checking OD 260/280 ratios, which was at 0.54.