Purification of iRFP Fluorescent Proteins

The iRFP702, iRFP713 and iRFP720 genes were cloned in the pBAD/His-B vector (Invitrogen)14 (link). The iRFP702, iRFP713 and iRFP720 proteins with polyhistidine tags on the N-termini were expressed in LMG194 bacterial cells (Invitrogen) bearing the pWA23h plasmid encoding heme oxygenase under the rhamnose promoter13 14 (link). To initiate protein expression, bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 5 h at 37 °C. Then 0.002% arabinose was added and bacterial culture was incubated for additional 12 h at 37 °C following by 24 h at 18 °C. Proteins were purified using Ni-NTA agarose (Qiagen). Ni-NTA elution buffer contained no imidazole and 100 mM EDTA. The elution buffer was substituted with PBS buffer using PD-10 desalting columns (GE Healthcare).