Pure fplc

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).

An aliquot of the tandem affinity purified complex was thawed and concentrated to 500 μL using Vivaspin 10 kDa cutoff PES-membrane concentrators (GE Healthcare) to avoid non-specific binding of the complex associated with cellulose membrane concentrators. The sample was loaded onto a S200 Increase 10/300 GL column (GE Healthcare) pre-equlibrated with PBS attached to a Akta Pure FPLC equipped with multiple wavelength monitoring at 280 nm and 488 nm. 0.5 mL fractions were collected, and fractions containing the main peak (generally 2.5 mL), corresponding to fully assembled sfGFP-A2:B subunit complexes as noted by co-incidence of the 280 nm peak and the 488 nm peak, were combined. For cell culture applications, the concentration of the complexes was determined by Abs485 using the reported molar attenuation coefficient of ε₄₈₅ 83300 M⁻¹ cm^{−1 46 (link)} (Supplementary Fig. 4); if the concentration was too low (<1 μM), the purified complexes were further concentrated.