| Marston FAO, Lowe PA, Doel MT,
(2005)
BIO/TECHNOLOGY,
2,
800-804 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| HB101 |
| 37.0 |
| not stated |
| pCT52 or pCT70 |
| Cells were grown in a 400L fermenter in medium at pH 6.5 containing per L: 3g KH2PO4, 6g Na2HPO4, 0.5g NaCl, 30g casamino acids, 4g NH4Cl, 0.022g CaCl2.H2O, 0.25g MgSO4.7H2O, 16g glycerol, 10mg thiamine, 0.1g carbenicillin, 0.5g proline, 1g yeast extract. no further details provided |
| Not Stated |
| OD =
|
| Sonication |
| Lysozyme |
| None |
| insoluble |
| Dilution |
| 50mM Tris pH 8.0, 10mM EDTA, 50mM NaCl, 0.1mM PMSF, 0..5% (v/v) Triton X-100 |
| 50mM Tris pH 8.0, 10mM EDTA, 50mM NaCl, 0.1mM PMSF, 0..5% (v/v) Triton X-100, 8M urea |
| 50mM KH2PO4, pH 10.7, 1mM EDTA, 50mM NaCl |
| None |
| yes |
| 10.7 |
| 25.0 |
|
| 1h |
| None |
| n/a |
| Harvested cells were resuspended in 3 volumes (wet weight/volume) of 50mM Tris pH 8.0, 1mM EDTA, 50mM NaCl, 0.1mM PMSF, then lysed using lysozyme/sodium deoxycholate by liquid shear or sonication. The cell lysate was centrifuged (12000g, 5min, 4degC) then suspended in 9 volumes (v/v) wash buffer, incubated at room temperature for 5min, then centrifuged.
The pellet was then suspended in solubilization buffer at a rate of 90ml buffer for every 10g wet weigh of starting material. After 1h, the solution was slowly added to 9 volumes (v/v) of refolding buffer and left for 30min. The pH was maintained at 10.7 with KOH. Finally the pH was adjusted to 8.0 with HCl and the extract was left at room temperature for at least 30min. The protein was then diafiltrated and purified by ion-exchange chromatography before activation of the mature protein. |
| SDS-PAGE |
| None |
| None |
|
|
|
|