| Hill S, Austin S, Eydmann T, Jones T, Dixon R.
(1996)
Biochemistry,
93,
2143-2148 |
| Undefined |
| C-terminal hexahistidine + tryptophan |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 0.0 |
| 00 |
| pJES409 |
| Echerichia coli strain BL21(DE3). Cultures were grown aerobically in Luria broth, and expression from the T7 promoter was induced by addition of 1 mM isopropyl /3-D-thiogalactopyranoside.
|
| IPTG |
| OD n/a =
n/a |
| Not stated |
| Lysozyme |
| Size-exclusion chromatography |
| insoluble |
| Dialysis |
| 50 mM Tris Cl, pH 8.5/300 mM NaCl/20 mM imidazole |
| 8 M ureaprotein, 50 mM Tris Cl, pH 8.5/300 mM NaCl/20 mM imidazole |
| 10 mM Tris Cl, pH 8/0.1 mM EDTA/1 mM dithiothreitol/5% glycerol/50 mM NaCl |
| Size-exclusion chromatography |
| no |
| 8.0 |
| 4.0 |
| n/a |
| overnight |
| DTT |
| 1 mM |
| Denaturation and Refolding of Histidine-Tagged NIFL.
Crude cell extract containing NIFL-6his was applied to a 5-ml chelating Superose column charged with NiCl2 in buffer A. Contaminating proteins were eluted by washing the column in the same buffer, and the purified NIFL remained attached to the column. The column was then washed with 8 M urea in buffer A, which removed the flavin from NIFL, and the denatured NIFL was then eluted from the column with 0.25 M imidazole in buffer A containing 8 M urea. The protein was refolded by dialysis into 10 mM Tris Cl, pH 8/0.1 mM EDTA/1 mM dithiothreitol/5% glycerol/50 mM NaCl overnight at 40C, and the material was further purified by gel filtration on Superose 12 to eliminate high molecular weight aggregates.
|
| Unspecified |
| None |
| Glycerol |
| 5% |
| n/a |
| n/a |
| n/a |