| Burgin, J., Schaller, J.
(1999)
Cell Mol Life Sci,
55,
135-141 |
| Crystallography,Structure-Function,Recombinant Protein Expression |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| M15 |
| 37.0 |
| 4.5h |
| pQE-8 |
| Cells were grown in 2x YT medium (100microg ampicillin/ml, 25microg kanamycin/ml). After induction the cells were harvested (4000g, 4degC, 30min). Protein was isolated by suspending in extraction buffer (6M guanidine hydrochloride, 0.1M sodium phosphate pH 8.0) This suspension was stirred overnight at 4degC and centrifuged for 30min(12,000g, 4degC). The supernatant was then loaded onto a Ni2+NTA-agarose column equilibrated with extraction buffer, pH 8.0 and pH 6.3 |
| IPTG |
| OD 600 =
0.7-0.9 |
| Chemical |
| Chemicals |
| Metal affinity chromatography |
| insoluble |
| Dilution/Dialysis combination |
| n/a |
| 6 M guanidine hydrochloride, 0.1 M sodium phosphate, pH 8.0, 5 mM DTT |
| 40 mM TrisHCl pH 8.0, 1.25 mM reduced and oxidized glutathione |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| n/a |
| DTT |
| 5mM |
| Solution containing the eluted protein was adjusted to pH 8.0 and DTT added. After overnight stirring at 4degC, protein was diluted 4x with refolding buffer over a 3h period. This solution was then stirred for an additional day at 4degC before being dialyzed against water for 4 days, lysophilized and desalted on a Sephadex G-15 column equilibrated with 50mM NH4HCO3. |
| Amino acid sequencing,Mass spectrometry |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |