| Boros, I. M., Tie, F., Giam, C. Z.
(1995)
Virology,
214,
207-214 |
| Protein-Protein Interaction Identification |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 2-3h |
| pET-BLV-His6 |
| Cells grown in LB medium containing 100microg/ml ampicillin. After induction, cells were harvested via centrifugation(10,000g, 10min, 4degC). The pellet was resuspended in 20ml of Buffer A (50mM Tris, pH 8.0, 1 mM EDTA, 100mM NaCl) containing 0.5mM PMSF. Cells were then lyzed and inclusion bodies collected by centrifugation(17,000g, 20min, 4degC). |
| IPTG |
| OD 600 =
0.6-0.8 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| PBS, pH 7.4, 1% Triton X-100, 1M GuHCl |
| PBS, pH 7.4, 6M GuHCl |
| 20mM HEPES, pH 7.9, 400mM KCl, 0.2 mM EDTA, 0.5mM PMSF, 0.5mM DTT and 20% glycerol |
| Metal affinity chromatography |
| no |
| 7.9 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| Inclusion bodies were washed 2x 20ml with washing buffer prior to solubilization and centrifugation. The solubilized supernatant was then mixed with 5ml Ni2+ chelating Sepharose Fast flow which had been preequilibrated with solubilization buffer. The column was washed with 20ml solubilization buffer containing 0.02 M imidazole and 15 ml of washing buffer. The protein was then eluted in PBS containing 0.3 M NaCl, 5% glycerol, and 0.25 M imidazole. Fractions containing protein were then dialyzed against refolding buffer. |
| Ligand Binding,Gel filtration chromatography |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |