| Couceyro PR and Fritz T
(32)
Protein Expression and Purification,
32,
185-193 |
| Recombinant Protein Expression |
| C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 37.0 |
| 5h |
| pET23b |
| 0.5L LB broth containing 34microg/ml chloramphenicol and 100microg/ml ampicillin was inoculated with 0.5ml of an overnight culture. Cells were grown at 37degC until OD600 reached 0.4-0.6. Expression was induced with 1mM IPTG and cells were incubated a further 5h, after which they were harvested by centrifugation (2000g, 10min, 4degC). |
| IPTG |
| OD 600 =
0.4-0.6 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| n/a |
| 6M GdnHCl, 0.5M NaCl, 20mM TrisHCl pH 8.0 |
| 50mM TrisHCl pH 8.0, 1mM GSH, 1mM GSSG, 1mM EDTA |
| Metal affinity chromatography |
| no |
| 8.0 |
| 25.0 |
| <0.1mg/ml |
| 7 days |
| GSH/GSSG |
| 1mM/1mM,1mM/1mM |
| Cells were lysed in solubilization buffer at 5ml buffer/g wet weight of bacterial pellet. Cells were sonicated for 15min on ice and centrifuged (20000g, 30min, 4degC). The supernatant was filtered, then loaded onto a Ni-NTA column with gentle shaking. The resin was washed with 20 column volumes each of solubilization buffer, urea buffer (8M urea, 0.5M NaCl, 20mM TrisHCl pH 8.0), solubilization buffer with 20mM imidazole and urea buffer with 20mM imidazole. The protein was then eluted with solubilization buffer containing 300mM imidazole.
The peptide was diluted to 0.5mg/ml and reduced in 150mM DTT, 4M GdnHCl, 50mM TrisHCl pH 8.0 for 4h, then refolded by dilution to less than 0.1mg/ml in refolding buffer and left gently shaking at room temperature for 7 days. Following refolding, the peptide was concentrated on a C18 reverse phase mini-column. |
| Bioactivity,Western Blot,HPLC,SDS-PAGE |
| None |
| None |
| n/a |
| n/a |
| 95% |
| A range of refolding buffers tested (see paper for more details). |