| Furuya K and Hutchinson CR
(1996)
Journal of Bacteriology,
178,
6310-6318 |
| Functional Studies |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3h |
| pET16b |
| Cells were grown in LB-medium at 26degC, 29degC or 37degC. When OD600 reached 0.6, 1mM IPTG was added and cells werer incubated a further 3h. Cells were centrifuged, and then resuspended in 1/10 culture volume of 50mM TrisHCl pH 7.5, 2mM EDTA, 0.1% Triton-X and 10% glycerol. |
| IPTG |
| OD 600 =
0.6 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| n/a |
| 1: PBS pH 7.5, 6M guanidine. 2: HEPES buffer pH 7.5, 6M urea |
| 1: PBS pH 7.5, 1mM MgCl2, 10mM DTT, 50% glycerol. 2:HEPES buffer pH 7.5, 50mM KCl, 10mM MgCl2, 10mM DTT, 50% glycerol |
| Metal affinity chromatography |
| no |
| 7.5 |
| 4.0 |
|
| 30h |
| DTT |
| 10mM |
| After lysis by sonication, the cell lysate was centrifuged (5min, 14000rpm). The cell pellet was reuspended in binding buffer containing 6M urea and centrifuged. The supernatant was passed down a Ni2+ affinity column, and the protein was purified under denaturing conditions (6m urea) as directed by the manufacturer (Novagen). The protein was precipitated by adding 2 volumes of H2O for later use
Refolding was performed in two buffer systems (1 and 2). In the eachh system, 1mg purified protein was resuspended in 10ml solubilization buffer (1 or 2) and dialyzed against the equivalent respective refolding buffer (1 or 2). Each sample was dialyzed three times against 100 volumes of buffer for 10h at 4degC. |
| DNA binding |
| None |
| None,Glycerol |
| 50% |
|
|
|