| Yamagata A, Masui R, Kakuta Y, Kuramitsu S, Fukuyama K
(2001)
Nucleic Acids Research,
29,
4617-4624 |
| Structure-Function |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 30.0 |
| 3h |
| pET19b |
| Cells were grown in 5L of 2xTY medium with 100microg/ml ampicillin at 30degC. When A600 reached 0.4, 0.4mM IPTG was added. Cells were grown for a further 3h then harvested by centrifugation and stored at -20degC |
| IPTG |
| OD 600 =
0.4 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| 20mM TrisHCl, 500mM NaCl, 10% glycerol, 5mM imidazole, 0.5mM PMSF pH 7.9 |
| 6M urea, 20mM TrisHCl, 500mM NaCl, 10% glycerol, 5mM imidazol pH 7.9 |
| 20mM TrisHCl, 0.1mM EDTA, 100mM NaCl, 10% glycerol pH 7.5 |
| Metal affinity chromatography |
| no |
| 7.5 |
| 4.0 |
| n/a |
| n/a |
| None |
| n/a |
| 10g frozen cells were thawed and resuspended in 80ml wash buffer then sonicated. The cell lysate was centrifuged (20000g, 15min) and the pellet was collected and washed with 80ml wash buffer. The protein was then extracted from the pellet in 80ml solubilization buffer and incubated at room temperature for 60min. After centrifugation (39000g, 60min), the supernatant was loaded onto a 5ml Ni-NTA column. The column was washed with 60ml solubilization buffer and the protein was eluted with 20ml solubilization buffer containing 100ml imidazole. The protein was then refolded by multi-step dialysis against solubilization buffer containing successively lower concentrations of urea (4,2,1M), followed by a final dialysis step against refolding buffer. The soluble protein was then collected by centrifugation (39000g, 60min) and purified further using a MonQ column. |
| Far-UV Circular Dichroism,SDS-PAGE,Gel filtration chromatography |
| None |
| Glycerol |
| 10% |
| 3mg/10g cellss |
| n/a |
| n/a |