Purification of mCherry-E20 Fusion Protein

mCherry plasmid (Addgene plasmid #54630) was digested with EcoRI/XhoI (NEB) and cloned into a pQE80 vector, modified in- house with glutamic acid tags (E-tag, E_n, where n = 20), as previously described,^{14 (link)} was transformed into competent BL21(DE3) E. coli and were grown in 3 × 1 L 2×YT media at 37 °C in a shaking incubator until OD₆₀₀ = 0.6, and then induced with 0.5 mM IPTG with shaking for a further 16 h at 16 °C. Cells were collected by centrifugation (3,200 g, 10 min, 4 °C), supernatant was discarded, and cells were resuspended in lysis buffer (50 mM Tris, 25 mM NaCl, 2 mM EDTA, pH 8 and were lysed by using 1% Triton X-100 (30 min, 37 °C, shaking) and then DNase-I (200 U mL⁻¹, 15 min, 37 °C, shaking) treatment. Lysed cells were then centrifuged (20,800 g, 30 min), the supernatant was collected and 150 mM NaCl was added. mCherry-E₂₀ was then purified using a HisPur cobalt column (Thermo Fisher) as per the manufacturer’s protocol. The column was washed with 1× PBS with 2 mM imidazole, eluted (150 mM imidazole, 50 mM NaH₂PO₄ and 300 mM NaCl), purified by dialysis in PBS (2 × 4 L, 10 kDa MWCO membrane), aliquoted and stored (−20 °C). Purity was checked using a 12% SDS-PAGE gel.