| Wu H, Gao J, Sharif WD, Davidson MK, Wahls WP
(2004)
Protein Expression and Purification,
38,
136-144 |
| Recombinant Protein Expression |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3h |
| pET15b+ |
| Cells were grown in 500ml LB medium containing 200microg ampicillin and 0.1% glucose. When A600 reached 0.6, expression was induced by the addition of 1mM IPTG and cells were incubated for a further 3h. Cells were then harvested by centrifugation, washed once with 15ml water and frozen as a cell pellet at -20degC. |
| IPTG |
| OD 600 =
0.6 |
| Sonication |
| Lysozyme |
| Metal affinity chromatography |
| insoluble |
| Dilution |
| 1:20mM TrisHCl pH 8.0, 0.1% Triton X-100, 1x protease inhibitors, 2:20mM TrisHCl, 500mM NaCl, 2M urea, pH 8.0 |
| 20mM TrisHCl, 100mM sodium phosphate, 6M GdnHCl pH 8.2 |
| 50mM TrisHCl pH 8.0, 250mM NaCl, 10mM KCl, 500mM L-arginine, 0.3mM lauryl maltoside, 400mm sucrose, 1mM EDTA, 2.5mM GSH, 0.25mM GSSG |
| Metal affinity chromatography |
| no |
| 8.0 |
| 4.0 |
| 0.4mg/ml |
| 16h |
| GSH/GSSG |
| 2.5mM/0.25mM,2.5mM/0.25mM |
| The cell pellet was thawed at 30degC for 15min, resuspended in wash buffer 1 containing 1mg/ml lysozyme and incubated at 30degC for 15min. The lysate was sonicated and then centrifuged (10000g, 30min, 4degC). The pellet was washed three times each at 22degC with 15ml wash buffer 1 and wash buffer 2. The final pellet was then dissolved in 15ml solubilization buffer and centrifuged. The supernatant was then stored at 22degC.
The protein was then purified using an Ni-NTA column and eluted with a gradient from the solubilization buffer to 20mM TrisHCl, 100mM sodium phosphate, 6M GdnHCl pH 4.5.
The protein was then diluted to 4mg/ml in elution buffer. The samples were adjusted to 10mM DTT and incubated at 60degC for 20min, then adjusted to 50mM iodoacetamide and incubated at 22degC for 45min. The protein was then diluted 40-fold in refolding buffer and incubated with gentle agitation for 16h at 4degC. The mixtures were then dialysed against a 50-fold excess of dialysis buffer (50mM TrisHCl pH 7.4, 150mM NaCl, 0.1mM EDTA, 1mM DTT, 10% glycerol) three times for 6h each at 4degC. The dialysate was then centrifuged (10000g, 30min, 4degC and the supernatant retained. |
| SDS-PAGE,Gel filtration chromatography |
| None |
| L-Arginine,Lauryl maltoside |
| 500mM/0.3mM |
| n/a |
| 95% |
| 1x protease inhibitors contains: 1 microg/ml aprotinin, 1 microg/ml leupeptin, 1 microg/ml tosylphenylalanine chloromethyl ketone, 1 microg/ml pepstatin A, 50microg/ml bestatin
A range of different refolding conditions tested, with varying compounds and protein concentrations, see paper for full details |