Pbad his b vector

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.

For crystallization, PCR-amplified BglII/EcoRI fragments encoding LSSmOrange, PSmOrange and PSmOrange2 were cloned into a pBAD/His-B vector (Invitrogen), modified by shortening the N-terminal polyhistidine tag to the MGSHHHHHHGRS- amino acid sequence. All proteins were expressed in LMG194 bacterial host (Invitrogen) grown in a minimal medium (RM) supplemented with 0.005% arabinose at 37°C for 24 h and at 25°C for 24 h upon shaking at 200 rpm. The cells were pelleted down by centrifugation, re-suspended in phosphate buffer saline, and lysed by sonication. The proteins were purified using Ni-NTA agarose (Qiagen) and size exclusion chromatography using Superdex 200 (16/60) column (GE Healthcare) followed by the dialysis against 10 mM NaH₂PO₄, pH 7.5.

The iRFP670, iRFP682 and iRFP713 genes were amplified and cloned into a pBAD/His-B vector (Invitrogen) using BglII and EcoRI sites. The iRFP670/C15S, iRFP670/C256S and iRFP670/C15S/C256S, iRFP682/C15S, iRFP682/C256S and iRFP682/C15S/C256S, iRFP713/T204A, iRFP713/C15S, iRFP713/C15S/V256C, iRFP713/V256C and iRFP713/C15S/V256C/T204A variants were obtained by site-directed mutagenesis using a QuickChange Mutagenesis Kit (Stratagene).
The iRFP670, iRFP682, iRFP713 proteins and their variants with polyhistidine tags on the N-termini were expressed in LMG194 host cells (Invitrogen) containing a pWA23h plasmid encoding heme oxygenase under the rhamnose promoter3 (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 cell culture was incubated for additional 12 h at 37 °C followed by 24 h at 18 °C. Proteins were purified using Ni-NTA agarose (Qiagen). The 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). The final purification was performed with ion-exchange chromatography using a MonoQ column (GE Healthcare).

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).

The BphP1-FP, iRFP670 and iRFP682 genes were cloned into the pBAD/His-B vector (Invitrogen). Site-specific mutagenesis was performed using QuikChange kit (Stratagene). The 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 promoter14 (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 followed 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). For spectroscopic experiments, the samples were diluted in PBS buffer composed of 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄ at pH 7.4.