| Tomasseli AG, Olsen MK, Hui JO, Staples DJ, Sawyer TK, Heinrikson RL, Tomich CC
(1990)
Biochemistry,
29,
264-269 |
| Drug Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BStl-c |
| 37.0 |
| 4-5h |
| pURA-PR3 |
| Cells were grown in LB medium with 0.2% glucose, 100microg/ml tryptophan, 100microg/ml ampicillin. An overnight culture was diluted 100-200-fold into LB broth with 100microg/ml ampicillin and grown at 30degC for 1h, then shifted to 37degC for 4-5h. |
| Temperature Shift |
| OD =
|
| Sonication |
| None |
| None |
| insoluble |
| Dilution |
| 10mM TrisHCl pH 7.4, 1mM EDTA |
| 6M GdnHCl |
| 50mM sodium acetate pH 5.5, 1mM EDTA, 2.5mM DTT, 10% glycerol, 5% ethylene glycol, 0.2% Nonidet P-40 |
| None |
| no tag |
| 7.5 |
| 4.0 |
|
|
| DTT |
| 2.5mM |
| Cells were harvested by centrifugation (10000g, 10min), resuspended in wash buffer and sonicated for 5min. The lysate was centrifuged (10000g, 10min), the pellet was then resuspended in wash buffer and resonicated. The inclusion bodies were then collected by centrifugation.
The inclusion bodies were then suspended in 200microL solubilization buffer, sonicated for 10min and placed on a a shaker for 60min at reoom temperature. The suspension was centrifuged (10000g, 10min) and the supernatant was diluted with 1volume of solvent B (0.15% TFA in 2-propranol/acetonitrile (7:3 v/v)) The protein was then purified by RP-HPLC.
The purified material was lyophilized, and the residue was dissolved at 5-15mg/ml in 50mM sodium acetate pH 5.5, 8M urea, 1mM EDTA, 2.5mM DTT. The protein was then diluted with 9 volumes of refolding buffer and allowed to stand at 4degC for a few minutes. |
| enzyme activity |
| None |
| None,Glycerol,Ethylene glycol |
| 5%/10% |
|
|
|