| Cantoni C, Ponassi M, Biassoni R, Conte R, Spallarossa A, Moretta A, Moretta L, Bolognesi M, Bordo D
(2003)
Acta Crystallographica Section D,
58,
1843-1845 |
| Crystallography |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| M15(pREP4) |
| 37.0 |
| 4h |
| pQE30 |
| Cells were grown by inoculating media with a 1:100 dilution of overnight culture. Cells were grown at 37degC until OD600 reached 0.7, at which stage expression was induced by addition of 1mM IPTG and incubation for a further 4h. |
| IPTG |
| OD 600 =
0.7 |
| Sonication |
| Lysozyme |
| None |
| insoluble |
| Dilution |
| 1: 50mM TrisHCl pH 8.0, 0.5% Triton-X 100, 100mM NaCl, 1mM EDTA, 1mM DTT/2: 2M urea, 2M NaCl, 50mM TrisHCl pH 8.0, 10mM DTT/3: 100mM TrisHCl pH 8.0, 2mM EDTA, 10mM DTT |
| 6M GdnHCl, 100mM TrisHCl pH 8.0, 10mM EDTA pH 8.0, 0.1mM DTT |
| 20mM TrisHCl pH 8.0, 0.5mM EDTA pH 8.0, 25% glycerol, 3mM reduced glutathione, 0.3mM oxidized glutathione |
| None |
| no tag |
| 8.0 |
| 25.0 |
|
| 72h |
| GSH/GSSG |
| 3mM/0.3mM |
| Cell pellets were recovered and resuspended in 100mM TrisHCl pH 8.0, 2mM EDTA pH 8.0, 10mM DTT, 0.5mg/ml lysozyme and left a 4degC overnight. The cells were then sonicated and washed consecutively with wash buffers 1, 2 and 3. The inclusion bodies were then dissolved in solubilization buffer with agitation for 48h at 4degC. Half of the supernatant was refolded in refolding buffer for 12h, then the remaining portion was added and left to refold for a further 48h. The refolded solution was then concentrated and dialyzed overnight at 4degC against 100mM NaH2PO4 and 10mM TrisHCl pH 8.0. The protein was purified further using Ni-NTA agarose followed by gel filtration chromatography using a HiPrep Sephacryl S100 column. |
| Crystallography |
| None |
| None,Glycerol |
| 25% |
|
|
|