| DAlessio KJ, McQueney MS, Brun KA, Orsini MJ, Debouck CM
(1999)
Protein Expression and Purification,
15,
213-220 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| LW29 |
| 37.0 |
| 3.5h |
| pET16b |
| Cells were grown in 1L LB medium with 50microg/ml ampicillin. When A650 reached 0.6, protein expression was induced with 1mM IPTG and cells were grown for a further 3.5h. Cells were collected by centrifugation (4200g, 30min) |
| Not Stated |
| OD 350 =
0.6 |
| Osmotic shock + sonication |
| None |
| None |
| insoluble |
| Dilution |
| 50mM Tris, 150mM NaCl, 5mM EDTA pH 8.0 |
| 50mM Tris, 150mM NaCl, 5mM EDTA, 8M urea, 10mM DTT pH 8.0 |
| 50mM K2HPO4, 5mM EDTA, 1mM reduced glutathione, 0.1mM oxidized glutathione, 0.7M L-arginine, pH 10.7 |
| None |
| no tag |
| 10.7 |
| 4.0 |
| 65microg/ml |
| overnight |
| GSH/GSSG |
| 1mM/0.1mM |
| The cell pellet from 1L culture was osmotically shocked by suspension into 50ml of 5mM Tris, 1mM EDTA, 20% sucrose, pH 8.0. After centrifugation (30min, 13000g), the pellet was dispersed using a tissuemizer into 25ml wash buffer and lysed by sonication (5x1min). The lysate was centrifuged, and the pellet was washed by another resuspension into 25ml wash buffer and centrifugation. The washed pellet was then solubilized by dispersion using a tissuemizer into 25ml Solubilization buffer. The solution was stirred at room temperature for 1hr then centrifuged (30min, 23000g) and filtered.
The protein was then refolded by a slow drop-by-drop dilution (1ml/min) into 85-fold volumes of refolding buffer which was stirred. After stirring overnight, the solution was concentrated and then centrifuged. The supernatant was dialyzed against 25mM Na2HPO4, pH 7.0 containing either 150mM or 500mM NaCl. |
| Enzyme activity, gel-filtration, infrared and fluorescence spectroscopy |
| None |
| None,L-Arginine |
| 0.7m |
|
| 95%+ |
|