Purification of Phytochrome Photoreceptors

BphP1 and BphP1-mRuby2 proteins with polyhistidine tags on the N-terminus were expressed in LMG194 bacterial cells (Life Technologies-Invitrogen) containing a pWA23h plasmid encoding heme oxygenase for biliverdin synthesis in Escherichia coli^{18 (link)}. The bacterial cells were grown in RM medium supplemented with ampicillin, kanamycin and 0.02% rhamnose for 6-8 h followed by an induction of the protein expression by adding of 0.002% arabinose. The proteins were purified using a Ni-NTA agarose (Qiagen). The PpsR2 and PpsR2-mVenus proteins with a Strep-tag-II at the N-terminus and a polyhistidine tag at the C-terminus was expressed in BL21(DE3) bacterial cells grown in LB medium supplemented with ampicillin for 6 h, followed by an induction of a protein expression with 250 μM IPTG. The proteins were first purified with a Ni-NTA agarose (Qiagen) followed by purification with a Strep-Tactin sepharose (IBA Lifesciences).