Purification of Mutant mCherry Proteins

The gene encoding mCherry was ligated into the plasmid pET30a with a C-terminal His-tag. Amino acid mutations were introduced by a modified QuikChange™ method [21 (link)]. The obtained pET30a-mCherry and pET30a-psRFP were transformed into E. coli BL21(DE3). The recombinant strains were grown in LB at 37 °C with shaking (210 rpm) until the absorbance at 600 nm (OD₆₀₀) reached about 0.6, and then 0.4 mM IPTG was added. The cells were further cultured at 25 °C with shaking (180 rpm) for 20 h. Cells were collected by centrifugation and broken open using a crusher SPCH-18 (Stansted Fluid Power Ltd.,London, UK). Protein purification was carried out with the nickel-nitrilotriacetic acid agarose resin (Invitrogen, New York, USA). SDS-PAGE analysis was performed to analyze the purity of finally obtained proteins. Buffer exchange of the purified proteins was performed with a PD-10 desalting column (GE Healthcare, New York, USA).