| Baneres, J.L., Mesnier, D., Martin, A., Joubert, L., Dumuis, A., Bockaert, J.
(2005)
J Biol Chem,
280,
20253-20260 |
| Drug Studies |
| N-terminal KSI sequence, C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| Rosetta(DE3)pLysS |
| 37.0 |
| 4h |
| pET31b |
| Transformed cells grown in 2YT medium containing 100mg/L and then at the same time cells induced 0.2 |
| IPTG |
| OD 600 =
1 |
| Sonication |
| None |
| Size-exclusion chromatography |
| insoluble |
| Dialysis |
| 1M urea, 25mM TrisHCl, pH 7.8, 500mM NaCL, 10microg/ml leupeptin, 10 microg/ml aprotinin and 0.5 mM phenylmethylsulfonyl fluoride |
| 25 mM TrisHCl, pH 7.8, 6 M urea, 0.2 (w/w) SDS, 0.2 2-mercaptoethanol |
| 6-0 M urea, 25 mM TrisHCl, pH 7.8, 0.2 (w/w) SDS, 0.2 2-mercaptoethanol |
| Size-exclusion chromatography |
| no |
| 7.8 |
| 25.0 |
| n/a |
| n/a |
| Beta-mercaptoethanol |
| 0.2 |
| Inclusion bodies were washed x2 in washing buffer, and solubilized. The solubilized proteins were loaded onto a Ni2+-NTA-agarose column. After washing with solubilization buffer, a linear imidazol gradient (0-200mM) in solubilization buffer was used to elute protein. The eluted fractions were then analysed by SDS page, and dialyzed against refolding buffer. Ketoisomerase fusion partner was removed via acid or cleavage with factor XA. To do this fusion protein was dialyzed in 50 mM TrisCl, 50mM NaCL, 1 mM CaCl2, 5mM lauryldimethylamino oxide, pH 6.5 and digested for 3hr at 20degC. Reaction stopped by addition of PMSF to a final concentration of 1 mM and reaction was dialysed against solubilisation buffer. |
| Far-UV Circular Dichroism |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |