| Chen G, Gouaux E
(1997)
Proc. Natl. Acad. Sci. USA,
94,
13431-13436 |
| Structural Studies,Recombinant Protein Expression |
| N-terminal poly-histidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 2h |
| pETGQ |
| Cells were grown at 37degC for 3h, expression was then induced with 1mM IPTG and cells were incubated for an additional 2h. Cells were harvested, then resuspended in 9ml of 50mM Tris HCl pH 8.0, 1mM EDTA, 100mM NaCl, 1mM PMSF with 10mg of lysozyme and 12mg of deoxycholate at 0degC for 20min, followed by incubation at 37degC for 10min. The suspension was cooled to ~24degC, and 60microL of 1M CaCl2 and 0.5mg DNaseI were added and the suspension was stirred at room temperature for 30min. The inclusion bodies were isolated by centrifugation (23000g, 4degC, 20min and were washed with 20ml ice-cold wash buffer 1, followed by 20ml ice-cold wash buffer 2 with 1mM PMSF.
The washed inclusion bodies were dissolved in solubilization buffer and the solution was centrifuged (125000g, 20degC, 30min). The supernatant was dialyzed against buffer E at 4degC overnight and then centrifuged (125000g, 4degC, 1h). The solubilized protein was then purified by size exclusion chromatography, cencentrated to 10mg/ml and stored at -80degC.
|
| IPTG |
| OD n/a =
n/a |
| Chemical |
| Lysozyme |
| Size-exclusion chromatography |
| insoluble |
| Dialysis |
| 1:50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl, 0.5% Triton X-100, 1mM PMSF, 2: 20mM TrisHCl pH 7.4, 1mM EDTA, 200mM NaCl |
| 50mM TrisHCl pH 7.4, 5mM EDTA, 8M GdnHCl, 5mM DTT |
| 250mM NaCl/KCl (25:1 NaCl:KCl), 1mM EDTA, 1mM DTT, 0.05% PEG 3350, pH 8.5 |
| Size-exclusion chromatography |
| no |
| 8.5 |
| 4.0 |
| 1.0mg/ml |
| 18h |
| DTT |
| 1mM |
| Cells were grown at 37degC for 3h, expression was then induced with 1mM IPTG and cells were incubated for an additional 2h. Cells were harvested, then resuspended in 9ml of 50mM Tris HCl pH 8.0, 1mM EDTA, 100mM NaCl, 1mM PMSF with 10mg of lysozyme and 12mg of deoxycholate at 0degC for 20min, followed by incubation at 37degC for 10min. The suspension was cooled to ~24degC, and 60microL of 1M CaCl2 and 0.5mg DNaseI were added and the suspension was stirred at room temperature for 30min. The inclusion bodies were isolated by centrifugation (23000g, 4degC, 20min and were washed with 20ml ice-cold wash buffer 1, followed by 20ml ice-cold wash buffer 2 with 1mM PMSF.
The washed inclusion bodies were dissolved in solubilization buffer at room temperature for 2-6h and the solution was centrifuged (125000g, 20degC, 30min). The supernatant was dialyzed against buffer 20mM NaOAc, 4M GdnHCl, 1mM DTT pH 4.5 at 4degC overnight and then centrifuged (125000g, 4degC, 1h). The solubilized protein was then purified by size exclusion chromatography, concentrated to 10mg/ml and stored at -80degC.
200microL of the protein was then refolded by dialysis against 20ml refolding buffer for 18h . The protein was then dialyzed against 40ml 100mM NaPhosphate pH 6.8, 200mM sodium sulfate, 10mM glutamate, 1mM DTT, 2mM EDTA at 4degC for 4h. The protein was then purified by gel filtration chromatography
|
| Ligand Binding,SDS-PAGE,Gel filtration chromatography |
| None |
| None |
| n/a |
| 20%, 20mg per L |
| 95% |
| 16 different refolding buffers screened (see paper for more details), refolding buffer noted here is the most effective buffer. Refolding by step-wise dialysis with serial reduction of GdnHCl concentration also performed but not as effective. |