Purification of BE3 Bacterial Protein

BE3 protein was prepared by overexpressing in BL21 Star (DE3)-competent E. coli cells using a plasmid encoding the bacterial codon-optimized base editor with a His₆ N-terminal purification tag. Detailed purification steps are described in our previous study^{11 (link)}, and the expression plasmid is available on Addgene (Note S1). After protein expression, bacteria cells were lysed by sonication and the lysate was cleared by centrifugation. The cleared lysate was incubated with His-Pur nickel nitriloacetic acid (nickel-NTA) resin. The resin was washed before bound protein was eluted with elution buffer. The resulting protein fraction was further purified on a 5 mL Hi-Trap HP SP (GE Healthcare) cation exchange column using an Akta Pure FPLC. Protein-containing fractions were concentrated using a column with a 100,000 kDa cutoff (Millipore) centrifuged at 3,000 g and the concentrated solution was sterile filtered through an 0.22 μm PVDF membrane (Millipore).