| Boucher, P. E., Maris, A. E., Yang, M. S., Stibitz, S.
(2003)
Mol Cell,
11,
163-173 |
| Protein-Protein Interaction Identification |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3h |
| pSS1744 |
| Induced cells were pelleted and washed with ice cold PBS, prior to resuspension in lysis buffer (20mM TrisHCl, pH 7.8, 20mM KCl, 5mM DTT, 5mM EDTA and 1mM PMSF. This mixture was then passed x2 through a French press at 16,000 lb/in2. The sample was then centrifuged (12,000g, 30min) and the supernatant discarded. |
| IPTG |
| OD 600 =
0.4-0.5 |
| French Press |
| Chemicals |
| Washing inclusion body |
| insoluble |
| Dialysis |
| 1. 10mM HEPES-NaOH, pH 7.4, 0.5M NaCl, 5mM EDTA, 1mM DTT, 1% Triton X-100. 2. 10mM HEPES-NaOH, 50mM NaCl, 5mM EDTA, 1mM DTT |
| 20mM MOPS, pH 8.0., 10mM MgCl2., 200mM KCl, 6M urea, 0.4mM EDTA |
| 20mM HEPES-NaOH, pH 7.4, 10mM MgCl2, 50mM KCl, 1mM DTT, 50% glycerol |
| Washing inclusion body |
| no |
| 7.4 |
| 4.0 |
| n/a |
| 48h |
| DTT |
| 1mM |
| Inclusion bodies were washed twice with washing buffer 1 and then once with washing buffer 2. Inclusion bodies were then resuspended in solubilisation buffer and centrifuged(13,000g, 10min) to remove insoluble material. The sample was then run through a Q-Sepharose Fast Flow column preequilibrated with column buffer. Purified BvgA was dialyzed against 10ml precipitating buffer(10mM HEPES-NaOH, pH 7.4, 50mM NaCl, 5mM EDTA, 1mM DTT) and precipitate collected by centrifugation(10,000g, 10min). Precipitate was resuspended to total protein concentration of 0.15mg/ml in 50mM NaH2PO4, pH 6.8, 6M GuHCl, 1 mM DTT and insoluble material removed by centrifugation, The sample was then dialyzed against several changed of refolding buffer over a 48 h period. |
| Ligand Binding |
| None |
| Glycerol |
| 50% |
| n/a |
| n/a |
| n/a |