Purification and Characterization of miRFP Proteins

The miRFP670 and miRFP709 genes were cloned into a pBAD/His-B vector (Invitrogen). Site-specific mutagenesis of miRFP670 was performed using QuikChange kit (Stratagene), resulting in miRFP670/C20A mutant. The miRFP670, miRFP670/C20A and miRFP709 proteins with polyhistidine tags on the N-termini were expressed in LMG194 bacterial cells (Invitrogen) bearing the pWA23h plasmid encoding a heme oxygenase under the rhamnose promoter^{5 (link)}. To initiate protein expression, the 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 the bacterial culture was incubated for an additional 12 h at 37 °C followed by 24 h at 18 °C. The resulting holoproteins were purified using a Ni-NTA agarose (Qiagen). An Ni-NTA elution buffer contained 100 mM EDTA and no imidazole. After elution, the buffer was substituted with phosphate-buffered saline using PD-10 desalting columns (GE Healthcare). In experiment with apoproteins, they were purified without co-expression of the heme oxygenase and then dissolved in 20 mM Tris-HCl buffer with 150 mM NaCl at pH or pD 8.0. The protein concentrations were ~0.45 and ~0.9 mM for time-resolved absorption and Raman experiments, respectively.