Expression and purification of bacteriorhodopsin with three mutations

We used the native halobacterial nucleotide sequence with two silent mutations (C9A, G12A corresponding to amino acids Ala2 and Gln3) as reported before⁶⁰ to express BR in E. coli. We appended to the native sequence of bacteriorhodopsin (BR, UniProt ID {"type":"entrez-protein","attrs":{"text":"P02945","term_id":"114811","term_text":"P02945"}}P02945) at the 3′ terminus additional GSGIEGRSGAPHHHHHHHH* tag, which was used for metal-affinity chromatography purification and contained FXa cleavage site, and introduced the gene into the pSCodon 2.1 expression vector (Delphi Genetics) via NdeI and XhoI restriction sites. The mutations T17A, T24A, and T47A were introduced by PCR. The protein was expressed and purified as described⁶⁰ with slight modifications. E. coli BL21(DE3) cells were transformed with pSCodon-derived plasmid and plated over LB-agar supplemented with ampicillin (0.05 mg/ml). The cells were grown at 37 °C in 2 L baffled flasks shaking at 120 rpm in an autoinducing medium ZYP-5052 containing ampicillin (0.05 mg/ml). At optical density OD₆₀₀ of 1.0 the temperature was decreased to 20 °C, and the media was supplemented with 10 μM all-trans-retinal and an additional portion of antibiotic. Another portion of antibiotic was added at OD₆₀₀ = 3.0. After 14 h of cultivation, the cells were harvested by centrifugation at 5000 rpm, resuspended in 20 mM Tris-HCl pH 8.0 supplemented with lysozyme (0.2 mg/ml) and DNAseI (0.02 mg/ml), and disrupted in M-110P Lab Homogenizer (Microfluidics, USA). Then 5 M NaCl was added to a final concentration of 200 mM and the suspension was layered over a glycerol cushion (3 ml—90% w/v) in 32 ml tubes. The total membranes were isolated by ultracentrifugation in SW-32Ti rotor (Beckman Coulter, Krefeld, Germany) at 28 000 rpm for 1 h, resuspended them in 20 mM Tris-HCl pH 8.0, 100 mM NaCl and solubilised overnight in DDM. The insoluble fraction was removed by ultracentrifugation (90,000 g, 1 h, 4 °C) and 10 mM of imidazole was added to the supernatant. Then it was loaded on a Ni-NTA column (Qiagen, Germany) and after washing the column with 5 volumes of 50 mM NaH₂PO₄/Na₂HPO₄ pH 8.0, 100 mM NaCl, 50 mM imidazole, 0.2% DDM and 1 volume of 50 mM NaH₂PO₄/Na₂HPO₄ pH 6.0, 100 mM NaCl, 50 mM imidazole, 0.2% DDM buffers we eluted the protein in a buffer containing 50 mM NaH₂PO₄/Na₂HPO₄ pH 6.0, 100 mM NaCl, 0.5 M imidazole and 0.3% DDM. Eluted protein applied to 24 ml Superdex 200i (GE Healthcare, Germany) column equilibrated with 50 mM NaH₂PO₄/Na₂HPO₄ pH 6.0, 100 mM NaCl, 0.2% DDM, and pooled a peak of coloured functional protein. Protein-containing coloured fractions were collected and concentrated to 60 mg/ml for crystallisation.