| Column refolding: Nickel-chelating chromatography |
| 20 mM PIPES, pH 7.4; 1.0 M urea; 1.0% (v/v) Triton X–100; 1 mM EDTA; 0.002% (w/v) PMSF |
| 4.0 M guanidinium–chloride and 100 mM DTT; 10 mM Tris–HCl, pH 8.0; 1 mM EDTA and 0.002% PMSF |
| 20 mM PIPES, pH 7.4; 1 mM EDTA; 0.1% Triton X–100, 0.002% PMSF |
| Washing inclusion body |
| no |
| 7.4 |
| 4.0 |
| 10 mg/ml |
| 48 h |
| DTT |
| n/a |
| Isolated inclusion bodies were solubilised in TEP buffer containing 4.0 M guanidinium–chloride and 100 mM DTT at a protein concentration of 10 mg/ml, and sonicating at 4 °C for 15 min. Insoluble material was removed by centrifugation for 15 min at 12 000 x g. Fresh Ni2+-NTA resin (1 ml) was washed in solubilisation buffer before being added to solubilised inclusion bodies (10 mg protein per ml resin). The slurry was stirred gently at room temperature for 30 min and then packed into a chromatography column. The column was washed at room temperature with 10 volumes of solubilisation buffer followed by 10 volumes of solubilisation buffer containing 50 mM imidazole, in order to remove contaminating proteins. Then the column was washed with 10 volumes of urea buffer (20 mM PIPES, pH 7.4; 1.0 M urea; 1.0% (v/v) Triton X–100; 1 mM EDTA; 0.002% (w/v) PMSF) followed by 10 volumes of refolding buffer (20 mM PIPES, pH 7.4; 1 mM EDTA; 0.1% Triton X–100, 0.002% PMSF). The column was cooled to 4 °C with the flow stopped, before being washed with a further 10 volumes of refolding buffer (4 °C). His-tagged OGC was eluted from the column by washing with folding buffer containing 0.3 M imidazole. Finally, the eluted OGC was dialysed against 20 mM PIPES, pH7.4; 0.1 M sucrose; 0.1% (v/v) Triton X-100; 1mM DTT and 0.002 % (w/v) PMSF. |
| Enzyme activity, gel-filtration, infrared and fluorescence spectroscopy |
| None |
| None |
| NULL |
| 1% |
| 99% |
|