| Dialysis |
| 20mM PBS pH 7.2, 5mM EDTA, 25% sucrose, 1% Triton X-100 |
| 7M urea, 10mM sodium phosphate pH 8.0, 10mM 2-ME, 2mM EDTA |
| 0.3M GdnHCl, 100mM sodium phosphate pH 7.4, 5mM 2-ME, 0.2mM EDTA |
| Ion-exchange chromatography |
| no tag |
| 7.4 |
| 4.0 |
| 0.7/3mg per ml |
|
| Beta-mercaptoethanol |
| 5mM,5mM |
| The cell pellet from 1.6L culture was washed with 70mL buffer P (20mM PBS pH 7.2, 5mM EDTA) and centrifuged (10min, 5000rpm) in two separate tubes. The cells were resuspended in 70ml ice-cold distilled water and kept on ice for 10min to allow for osmotic swelling. After centrifugation (10min, 10000rpm), the pellet was resuspended in 20ml buffer P containing 10U of bnzone nuclease. The suspension sonicated on ice, then more buffer P was added (up to about 30ml each tube), and the mixture was incubated for 30min at 37degC to digest nucleic acids. The lysate was then centrifuged (4degC, 12000rmp, 15min). The pellet was resuspended in 15ml wash buffer and sonicated for 1min. 30ml wash buffer was added to each tube, and the inclusion bodies were centrifuged again (10min, 160000rpm). This process was repeated twice, then the pellets were washed in distilled water. The inclusion bodies were then dissolved in 15ml solubilization buffer by stirring overnight at room temperature.
The protein fragments were purified by cation exchange chromatography on an FPLC Resources column at 4degC using solubilization buffer for binding and washing. The protein was applied to the column in 2ml aliquots and was eluted with 150mM NaCl in solubilization buffer.
Proteins were refolded by dialysis against refolding buffer at 4degC. |
| Far-UV Circular Dichroism |
| None |
| None |
|
|
|
| Several different refolding buffers tried. More success with refolding with aa.168-380 fragment together under very specific conditions (see paper for more details). Alternatively, bette yields again achieved by co-expressing the two fragments together in a soluble form. |