| Dilution |
| 25mM HEPES, pH 7.5, 300 mM NaCl and 300 mM Imidazole |
| 25mM HEPES, pH 7.5, 300 mM NaCl |
| 25mM Tris, pH 7.5, 200mM guanidine chloride, 4mM GSH, 0.4mM GSSG and 0.1% NaN3) |
| Metal affinity chromatography |
| yes |
| 7.5 |
| 25.0 |
| 10 uM |
| 4 days |
| GSH/GSSG |
| n/a |
| The bacteria were collected and resuspended in 100 ml HS300 buffer (25mM Hepes, pH7.5, 300mM NaCl). After ultrasonication treatment, bacteria were ultracentrifuged at 235,000g for 1 h and the supernatant was loaded onto a 5 ml Hitrap Ni-chelating column (Pharmacia) that was prepared according to the procedure outlined in the manufacturer’s manual and pre-equilibrated with HS300 buffer. The column was extensively washed with HS300 buffer containing 25mM imidazole. The fusion protein was eluted by HS300 buffer with 250mM imidazole.
Unfolding of the fusion protein was performed by overnight dialysis against 25mM Tris buffer, pH 7.5, 5mM DTT. The sample was next diluted with refolding buffer (25mM Tris, pH 7.5, 200mM guanidine chloride, 4mM GSH, 0.4mM GSSG, and 0.1% NaN3) to final concentration of 10 uM. The resulting solution was capped with air and kept at room temperature. The crude refolded product was recollected following passage through a 1 ml Hitrap Ni-chelating column and purified by the same protocol as outlined above.
The digestion of the fusion protein was done overnight using 200U of thrombin, while dialyzing against 25mM Tris buffer, pH 7.5, at 4 degree. Finally, the sample was loaded onto Resource S column (Pharmacia) and then eluted using a NaCl gradient from 0 to 800mM.
|
| 15N -1H chemical shifts (ppm) |
| None |
| None |
| NULL |
| < 20% |
| NMR grade |
|