| Calderone, V., Trabucco, M., Menin, V., Negro, A., Zanotti, G.
(2002)
Biochim Biophys Acta.,
1596,
283-292 |
| Recombinant Protein Expression |
| N-terminal hexahis + Xpress tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 20.0 |
| overnight |
| pRSETB |
| Cells were grown at room temperature, induced and allowed to incubate overnight. Cells were then harvested by centrifugation(6000rpm, 20min) and washed in PBS buffer (140mM NaCl, 2.7 mM KCl, 10mM Na2PO4, pH 8.0) and resuspended in 20mM TrisHCl, 50mM NaCl, pH 8.2. Cells were then lyzed and ultracentrifuged(12,000rpm, 45min) to isolate inclusion bodies and membranes. Material was then applied to a Ni-NTA column. |
| IPTG |
| OD 595 =
0.6 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 100mM NaH2PO4, 10mM TrisHCl, 8M urea, pH 8.0 |
| 50mM TrisHCl, 200mM NAcCl, 10mM beta-mercaptoethanol, pH 8.2 |
| Metal affinity chromatography |
| yes |
| 8.2 |
| 4.0 |
| n/a |
| 4days |
| Beta-mercaptoethanol |
| 10mM,10mM |
| 3-HAO eluted from the Ni-NTA column with 40-150mM imidazole was analyzed by sDS page. Inclusion bodies were then suspended in solubilization buffer with stirring at room temp for 1h. The sample was then ultracentrifuged (12,000rpm, 1h) to separate from membranes. Denatured protein was applied to IMAC column and washed with solubilization buffer, before elution via lower pH. Refolding took place on fraction that had been eluted at pH 4.5 by dialysis against refolding buffer. |
| Far-UV Circular Dichroism,Enzyme activity,Kinetic analysis |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |