| Dilution |
| 10mM TrisHCl pH 7.5, 1mM EDTA, 1M NaCl |
| 100mM Tris-HCl, 0.2mM EDTA, 6M GuHCl, pH 7.5 |
| 100mM Tris-HCl, 0.5 L-arginine, 0.2 EDTA, pH 7.5 |
| None |
| no |
| 7.5 |
| 10.0 |
|
| 36-48 |
| None |
| n/a |
| The cell pellet (wet weight ~3-3.5g) was washed with ~300ml buffer A (10mM TrisHCl pH 7.5, 10mM EDTA, 100mM NaCl) then resuspended thoroughly in the same buffer containing 1mM PMSF. The cells were then lysed by sonication, the lysate was then stirred for 1h at 4degC with an equal volume of Buffer A containing 8M urea and 1mM PMSF. The lysate was then centrifuged (45min x 10000rpm x 4degC) and the pellet, containing the inclusion bodies, was then washed once with Wash buffer and once with distilled water before undergoing centrifugation again.
The inclusion bodies were then resuspended in ~10ml solubilization buffer, stirred on a magnetic stirrer for ~2h at reoom temperature and then centrifuged. The concentration of protein in the supernatant was deteremined by Bradford assay. The solubilized inclusion bodies were then diluted with solubilization buffer to a final concentration of 10mg/ml. Refolding was initiated by rapid dilution of the solubilized inclusion bodies into freshly prepared refolding buffer prechilled to 10degC. Generally, 6ml solubilized inclusion bodies were added rapidly to 0.6L refolding buffer which was stirred. Two further additions (6ml) each were made at 1.5h intervals, the refolding reaction was allowed to incubate for 36-48h at 10deg C without stirring. The refolded protein was then dialyzed for 48h against 20mM phosphate buffer(pH 7.5) with 300mM NaCl, 10mM imidazole andd 100mM freshly prepared urea with buffer changes every 12h. The dialysed sample was centrifuged and filtered (0.45 micron).
The protein was then loaded onto a Ni-NTA matrix (~10ml packed volume, which was then washed extensively with equilibration buffer (20mM phosphate pH 7.5, 300mM NaCl, 10mM imidazole) with 100mM urea prior to overnight incubation at 4degC with gentle mixing. The resin was then packed into a column and the flow-through collected. The column was washed with 50ml equilibration buffer with 20 mM imidazole and eluted sequencially with 100, 250 and 500mM imidazole-containing equilibration buffers. Selected fractions were pooled and dialyzed against PBS. |
| Virus Binding Blocking assay |
| None |
| None,L-Arginine |
| 0.5M |
| 30mg/L |
|
| Different concentrations of L-arginine used in smaller scale experiments to find optimal conditions (see paper for details) |