| Cipakova I, Gasperik J, Hostinova E
(2006)
Protein Expression and Purification,
45,
269-74 |
| Recombinant Protein Expression |
| N-terminal KSI sequence, C-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BLR(DE3)pLysS |
| 37.0 |
| 5h |
| pET31b |
| Cells were grown in LB medium containing chloramphenicol and ampicillin at 37degC. When OD600 reached 0.5, 1mM IPTG was added and cells were incubated a further 5h. Cells were harvested by centrifugation (8000g, 4degC, 15min) |
| IPTG |
| OD 600 =
0.5 |
| Sonication |
| None |
| Metal affinity chromatography |
| insoluble |
| Dialysis |
| 20mM TrisHCl pH 7.9, 0.5M NaCl, 5mM imidazole |
| 20mM TrisHCl pH 7.9, 0.5M NaCl, 5mM imidazole, 6M GdnHCl |
| dH20 |
| Metal affinity chromatography |
| yes |
| 7.0 |
| 4.0 |
| n/a |
| o/night |
| None |
| n/a,n/a |
| 10g frozen cells was resuspended in 20ml wash buffer and lysed by sonication. The The lysate was centrifuged (9000g, 4degC, 45min) and the pellet was washed in 50-100ml wash buffer and centrifuged again (5000g, 4degC, 15min). The inclusion bodies were dissolved in 20ml solubilization buffer by stirring for 1h at 0degC and the solution was centrifuged again. The supernatant was loaded onto a nickel-charged His Bind column, which was then washed extensively with solubilization buffer containing 5mM, then 60mM imidazole. The protein was then eluted in solubilization buffer containing 1M imidazole. Selected fractions were pooled and dialyzed overnight at 4degC against dH2O.
The precipitated fusion protein was then pelleted (8000g, 4degC, 5min) and treated with 2mg CNBr/mg fusion protein (in 80% formic acid, 22-24h at 25degC). The cleaved protein was then diluted 10-fold with ionized water and evaporated at 28degC using rotary evaporator. The protein was the repeatedly extracted to 100microL dH2O. |
| Bioactivity,Mass spectrometry |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |