| Column refolding: Size-exclusion chromatography |
| 20mM TrisHCl, 30mM NaCl pH 7.5 |
| 6M GdnHCl pH 5.5, 0.01mM GSH, 0.002mM GSSG |
| 10mM sodium acetate pH 5.0 |
| None |
| no tag |
| 5.0 |
| 22.0 |
| 1.38mg/ml |
|
| None |
| n/a |
| Cells were disrupted using a high-pressure homogenizer. The homogenate was centrifuged to collect the inclusion bodies. The inclusion bodies were suspended in 500ml wash buffer and centrifuged again (15min x 8000g). The pellet was then resuspended in 500ml of 10mM EDTA, pH 5.5 and stored at -80degC until use. The inclusion bodies were solubilized in 12L 6M GdnHCl pH 5.5 and allowed to stand for 2h at 22degC. 0.01mM GSH and 0.002mM GSSG were then added to the solubilized protein and the supplemented solution was adjusted to pH 8.5 with 10mM TrisHCl and incubated for 16h at 22degC. The oxidation yield was then confirmed by reverse-phase HPLC analysis, then the solution was applied to a gel filtration column (Sephadex G-25 M) equilibrated with refolding buffer. The protein was eluted with the same buffer at 750ml/min.
The refolded protein was then adjusted to 250mM sodium acetate pH 5.0 and applied to a cation=exchange column (CM-Sepharose FF) equilibrated with 10mM sodium acetate pH 5.0.The column was washed with 1 column volume(CV) of the same buffer, then the protein was eluted with a linear gradient of sodium acetate from 10mM to 500mM and a pH gradient from 5.0 to 5.5 over 10CV at a flow-rate of 0.5L/min.
Pooled fractions were adjusted to pH 4.0 with formic acid and applied to a preparative reverse-phase HPLC column equilibrated witn 0.1M sodium formate pH 4.0.The column was washed with 1CV of the same buffer, then the protein was eluted with a linear gradient of 0-60% acetonitrile in 0.1M sodium formate over 6CV at 50ml/min. Fractions were then applied to a gel filtration column (Sephadex G-25M)equilibrated with with 20mM acetic acid and 10% acetonitrile and the proteins were eluted with the same solvent at 120ml/min, kept at 22degC for 1h, then applied to another Sephadex G-25M column with 5mM sodium acetate pH 4.5 at 350ml/min. The protein was then concentrated to >8mg/ml by cation-exchange chromatography using a CM-sepharose FF column, then the protein was applied to a HPLC Superdex-75 column equilibrated with 10mM sodium citrate pH 6.0 and el;uted in the same buffer at 24ml/min
|
| HPLC |
| None |
| None |
|
|
|
|