The RpBphP6 and iRFP mutants with polyhistidine tags on the N-terminus were expressed in LMG194 bacterial cells grown in RM medium supplemented with ampicillin, kanamycin, 0.002% arabinose, 0.02% rhamnose for 15-18 h at 37°C and then for 24 h at 18°C. Proteins were purified using Ni-NTA agarose (Qiagen) according to the manufacturer’s protocol with minor modification. In wash buffer 400 mM imidazole was substituted with 100 mM EDTA. Fluorescence spectra were recorded using a FluoroMax-3 spectrofluorometer (Jobin Yvon). A Hitachi U-2000 spectrophotometer was used for absorbance measurements.
Extinction coefficient was calculated based on a comparison of absorbance values at the main peak with the absorbance value at the smaller peak at around 390 nm, assuming the latter had extinction coefficient of free BV of 39,900 M−1cm−1. To determine quantum yield, fluorescence signal of a purified protein was compared to that of an equally absorbing Nile Blue dye. pH titrations were done using a series of buffers (100 mM sodium acetate, 300 mM NaCl for pH 2.5–5.0 and 100 mM NaH2PO4, 300 mM NaCl for pH 4.5–9.0).
To study protein folding and maturation in cells21 (link), LMG194 bacterial cells were grown at 37°C overnight in RM medium. The next morning, 0.2% rhamnose was added for 2 h, then 0.002% arabinose was added, and cells were cultured for 1 h. Then arabinose was washed out and cells were cultured in RM medium with 0.2% rhamnose at 37°C. Over time fluorescence intensities of the equal aliquots of the cell suspension were measured after dilution to the same optical density of 0.2, and the obtained values were multiplied by the dilution factor. For EGFP expression, the procedure was the same except that LMG194 strain did not contain pWA21h plasmid for heme oxygenase expression.
Extinction coefficient was calculated based on a comparison of absorbance values at the main peak with the absorbance value at the smaller peak at around 390 nm, assuming the latter had extinction coefficient of free BV of 39,900 M−1cm−1. To determine quantum yield, fluorescence signal of a purified protein was compared to that of an equally absorbing Nile Blue dye. pH titrations were done using a series of buffers (100 mM sodium acetate, 300 mM NaCl for pH 2.5–5.0 and 100 mM NaH2PO4, 300 mM NaCl for pH 4.5–9.0).
To study protein folding and maturation in cells21 (link), LMG194 bacterial cells were grown at 37°C overnight in RM medium. The next morning, 0.2% rhamnose was added for 2 h, then 0.002% arabinose was added, and cells were cultured for 1 h. Then arabinose was washed out and cells were cultured in RM medium with 0.2% rhamnose at 37°C. Over time fluorescence intensities of the equal aliquots of the cell suspension were measured after dilution to the same optical density of 0.2, and the obtained values were multiplied by the dilution factor. For EGFP expression, the procedure was the same except that LMG194 strain did not contain pWA21h plasmid for heme oxygenase expression.