| Dilution with complex partner subunits |
| 50mM TrisHCl, 1%(v/v) Triton X-100, 100mM NaCl, 1mM sodium EDTA, 0.1% sodium azide, 1mM DTT pH 8.0 |
| 25 2-(N-morpholino)ethanesulfonic acid, 8M urea, 10mM sodium EDTA, 0.1mM DTT pH 6.0 |
| 100mM TrisHCl, 1M L-arginineHCl, 2mM sodium EDTA, 6.3mM cysteamin(2-mercaptoethylamine), 3.7mM cystamine, 0.5mM PMSF pH8.5 |
| None |
| yes |
| 8.5 |
| 10.0 |
|
| 42-60h |
| cysteamine/cystamine |
| n/a |
| 6L LB media containing 0.4% (w/v) glucose, 30mg/L chloramphenicol and 100mg/L ampicillin was inoculated with a single bacterial colony and grown until OD600 reached 0.7-1.0. 0.5mM IPTG was added and cells were grown for a further 2h before being harvested. The cell pellets were suspended in a buffer containing 50mM TrisHCl, 25% sucrose, 1mM sodium EDTA, 0.1% sodium azide and 10mM DTT pH 8.0. 1mg/ml lysozyme, 25microg/ml DNaseI and 5mM MgCl2 were added. Lysis buffer (2.5ml per ml cell suspension) containing 50mM TrisHCl, 1%(v/v) Triton X-100, 1% (sodium deoxycholate, 100mM NaCl, 0.1% sodium azide and 10mM DTT pH 8.0 was added to the cells. AFter the viscosity decreased, 10mM sodium EDTA was added and the suspension was frozen and then thawed. The cell lysate was centrifuged, and the pellets were reuspended and centrifuged in wash buffer four times. The inclusion bodies werer then suspended in 50mM TrisHCl, 1mM sodium EDTA, 0.1% sodium azide and 1mM DTT pH 8.0, centrifuged and dissolved in 10-20ml solubilization buffer and ultracentrifuged (140000g). Aliquots of the supernatant were stored at -70degC.
45mg of TCR-A6-alpha protein and 30mg of TCR-A6-beta protein, both dissolved in 8M urea were added together to approximately 15ml of 6M GdnHCl, 10mM sodium acetate and 10mM EDTA pH 5.2 at room temperature. 1L of refolding buffer was cooled to 10deg. The 15ml GdnHCl refolding solution containing alpha and beta subunits was diluted into the refolding buffer with vigorous stirring. The refolding solution was incubated at 10degC for 6-12h, then a second 15ml mixture of alpha and beta subunits was added, followed by a third 15ml aliquot another 6-12h later. Incubation continued at 10degC for another 24h. The refolding buffer was then dialyzed against 10L of 100mM urea at 4degC for 24h and then against 10L 100mM TrisHCl , 100mM urea pH 7.5 for another 24h. 0.5mM PMSF was added and the protein was purified further using a DE52 anion exchange column.
|
| Native gel band-shift assay |
| None |
| None |
| NULL |
|
|
|