| Adams JJ, Jang CJ, Spencer HL, Elliott M, Smith SP
(2004)
Protein Expression and Purification,
38,
258-263 |
| NMR |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 4h |
| pET21b |
| Cells were grown in 1L LB mdia containing 100 micrograms/ml ampicillin at 37degC with shaking to an OD600 of 0.6. Protein expression was induced with 1mM IPTG and growth was continued for a further 4 hours. |
| IPTG |
| OD 600 =
0.6 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 25mM TrisHCl pH 7.4, 250mM NaCl, 8M Urea |
| 25mM TrisHCl pH 7.4, 50mM NaCl, 10mM EDTA |
| Metal affinity chromatography |
| no |
| 7.4 |
| 4.0 |
|
|
| None |
| n/a |
| Cells were pelleted and resuspended in a volume of resolubilization buffer ten times their packed weight and then sonicated at room temperature. Following centrifugation (1h x 40000rpm), the supernatant was applied to a column containing 2ml nickel chelating resin equilibrated with solubilization buffer. The column was washed with 20ml solubilization buffer containing 20mM imidazole, then protein was eluted with solubilization buffer containing 300mM imidazole in 1ml fractions. Suitably pure fractions were selected by SDS-PAGE analysis and pooled. The pooled fractions were refolded by dialysis in refolding buffer. The refolded protein was then applied to a Superdex gel filtration column and eluted in 2ml fractions in refolding buffer. Suitable fracters were pooled and concentrated to a final volume of 5ml. |
| 15N -1H chemical shifts (ppm) |
| None |
| None |
|
| 40mg/L in LB broth |
|
| Also grown in M9 minimal media for NMR analysis (yield 25mg/L) |