| Braspenning, J., Manetti, R., Zumbach, K., Meschede, W., Gissmann, L., Tommasino, M.
(1997)
Protein Expression and Purification,
10,
192-201 |
| Recombinant Protein Expression |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Schizosaccharomyces pombe |
| leu1-32h |
| 30.0 |
| 5-6 days |
| L20-E7 |
| Following reaching a density of approximately 2x10^7 cells, yeast cells were harvested and washed 3x with ice cold buffer R(25mM TrisHCl, pH 8.0, 10% glycerol, 20mM EDTA, 2mM DTT, 20mM NaF, and 0.1mM Na-o-Vanadate). An equal amount of cold acid-washed glass beads was then added and the pellet was disrupted using a bead beater. The supernatant containing the cell extract was centrifuged(20min, 12,000rpm, 0degC) and the resulting pellet resuspended in buffer R containing 5% Triton using a Dounce homogenizer and centrifuged. This process was repeated twice to obtain the recombinant protein. The E7 protein was then extracted from the pellet using Buffer AR (Buffer R without EDTA and containing 6M guanidinium hydrochloride). The extract was centrifuged(200rpm, 10min, 0degC). |
| Not Stated |
| OD n/a =
n/a |
| Bead Beater |
| None |
| Hydroxyapatite Chromatography/Ion-Exchange Chromatography |
| insoluble |
| Dialysis |
| n/a |
| 10mM Na-phosphate, pH 7.5, 10% glycerol, 2mM DTT, 8M Urea |
| 5mM Hepes, pH 7.2, 5% glycerol, 1mM DTT, 20microM Zn-acetate |
| Hydroxyapatite Chromatography/Ion-Exchange Chromatography |
| no |
| 7.2 |
| 20.0 |
| n/a |
| n/a |
| DTT |
| 1mM |
| Supernatant from AR buffer treaded cell extract underwent 12 fold dilution with solubilization buffer, The diluted sample was applied to a hydroxyapatite column which had been pre-equilibrated with solubilization buffer. Samples were washed with 5 column volumes of solubilization buffer with a 10% addition of buffer B1 (150mM Na-phosphate, pH 7.8, 10% glycerol, 2mM DTT, 8M urea, and 1 M NaCl). Recombinant E7 was eluted using a linear increasing gradient of B1. Fractions containing E7 were pooled and dialyzed overnight at 4degC against buffer A2 (50mM TrisHCl, pH 7.5, 0.1 mM Zn-acetate, 1 mM DTT, 4 M urea and 50mM NaCl). This required three buffer changes. The fractions were loaded onto a Q-Sepharose column which was equilibrated with buffer A2 and after washing with 5 columns worth of buffer A2. E7 was then eluted with increasing gradient from 0-75% of B2. Fractions then underwent Gel electrophoresis and fractions with pure E7 were dialyzed to refold the protein using 50 volumes refolding buffer. |
| Enzyme activity,Ligand Binding |
| None |
| Glycerol |
| 5% |
| n/a |
| n/a |
| n/a |