| Thies MJW, Pirkl F
(2000)
J Chromatography B,
737,
63-69 |
| Folding |
| C-terminal polar/charged tail |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| HB101 |
| 37.0 |
| 4h |
| pAkF-T5 |
| Cells were grown in SB medium (per L: 20g bactotryptone, 10g yeast extract, 5g NaCl, 2.5g K2HPO4, 1g MgSO4.7H2O) containing ampicillin and kanamycin at 37degC, expression was induced with 1mM IPTG. After 4h, cells were harvested by centrifugation (4000g). |
| IPTG |
| OD =
|
| High pressure homogenization |
| Lysozyme |
| None |
| insoluble |
| Dilution |
| 100mM TrisHCl, 20mM EDTA pH 7.0 |
| 100mM Tris, 6M GdmCl, 100mM 1,4-dithioerythrit (DTE), pH 8.0 |
| 0.7M TrisHCl, 2mM EDTA, 5mM GSSG, pH 8.0 |
| None |
| yes |
| 8.0 |
| 10.0 |
| 200microgram/ml |
|
| GSSG |
| 5mM |
| Cells were resuspended in 100mM TrisHCl, 1mM EDTA, pH 7 at 4degC then incubated with lysozyme (1.5mg/g cells) for 30min at 4degC before disruption by high-pressure treatment. DNA was removed by the addition of 10microgram/ml DNAseI and 3mM MgCl2 and incubation for 30min at room temperature. Then half a volume of 60mM EDTA, 6% TritonX-100, 1.5M NaCl pH 7.0 was added and the mixture was incubated a further 30min at 4degC. The mixture was centrifuged (40000g) and the pellet was washed with wash buffer.
The pellet was then resuspended in solubilization buffer at a ratio of 1-2ml buffer per 50mg pellet After 2hr of incubation at 25degC the pH was adjusted to 2 with 1M HCl. The mixture was then centrifuged (40000g) and the supernatant was dialyzed against 4M GdmCl pH2 at 4degC and the protein concentration was determined.
The protein was then refolded at 10degC by step-wise addition of the unfolded protein to refolding buffer at each hour. The final protein concentration was 200microgram/ml.
Ammonium sulfate was added to the refolded protein solution until a final concentration of 1.5M was reached. The refolded protein was then loaded onto a butyl-sepharose column equilibrated with 100mM TrisHCl, 2mM EDTA, 1.5M ammonium sulfate pH 7.0. The protein was eluted from the column by a linear gradient from 1.5 to 0M ammonium sulfate.
The protein was then passed down a Superdex75 prep grade column at 4degC, the buffer used was 100mM TrisHCl, 2mM EDTA, 300mM NaCl pH 7.0 |
| Fluorescence |
| None |
| None |
|
| 25% |
|
|