| Edwards GM, Huber HE, DeFeo-Jones D, Vuocolo G, Goodhart PJ, Maigetter RZ, Sanyal G, Oliff A, Heimbrook DC
(1992)
J Biol Chem,
267,
7971-7974 |
| Structure-Function |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 2 |
| pTAC |
| Cells were grown at 37degC in 3xLS medium plus 100mg/L apicillin until A600 reached 5. 1mM IPTG was added and cells were grown for a further 2h. The cell paste, about 2kg from 200L was frozen and stored at -70degC. |
| IPTG |
| OD 600 =
5 |
| Sonication |
| Lysozyme |
| None |
| insoluble |
| Dilution |
| 6M urea, PBS (6mM sodium phosphate, 150mM NaCl pH 7.2), 1% Triton |
| 50mM TrisCl pH 8.0, 50mM NaCl, 1mM EDTA, 6M GdnHCl |
| 0.1M TrisCl, pH 8.0, 0.4M MgCl2, 0.6M GdnHCl, 0.1mM EDTA, 0.1mM DTT, |
| None |
| no tag |
| 8.0 |
| 4.0 |
|
| 5h |
| DTT |
| 0.1mM |
| 200g of cell paste was reuspended in phosphate-buffered saline (PBS) containing 0.1mM PMSF, 1mM MgCl2, 0.01mg/ml DNaseI and 0.1mg/ml lysozyme at 2.5ml/g cell paste. The mixture was stirred for 30min and the cells were lysed by sonication. An additional 0.5volume of PBS was added and the mixture was centrifuged (20min, 13000g). PBS and solid urea were added to the pellet to give a final urea concentration of 6M and a volume of 3ml/g cell paste. Triton X-100 was added to 1% and the mixture was stirred for 1h.
After dilution with 1 volume of PBS, the solution was centrifuged (20min, 13000g). The pellet was washed with PBS and then reususpended in 50mM TrisCl pH 8.0, 50mM NaCl, 1mM EDTA. solid GdnHCl was added to give a final concentration of 6M and approximately 2ml/g cell paste. Following a brief incubation, the solution was centrifuged (20min, 13000g) and the supernatant was frozen.
Purification was typically performed on a 10ml aliquot of the guanidine extract (from approx 7g cell paste). Solid DTT was added to 0.1M, and the mixture was stirred at 30degC for 30min. Refolding was performed by dropwoise 100-fold dilution of the extract into 1L of refolding buffer containing a protease inhibitor mixture (10microg/ml benzamidine, 5microg/ml leupeptin, pepstatin A and aproptinin). The refolding mixture was stirref for at least 5h, then centrifuged (60min, 10000g). The buffer was changed to 50mM HEPES pH 7.0, 250mM NaCl, 0.1% Nonidet P-40 and 1mM DTT by cross-flow filtration. The dialysate was centrifuged (1h, 11000g) and then filtered. The filtrate was then purified further by passage through a 2ml ethanolamine-capped AffiGel 15 guard column and a 2ml E7-(20-29) peptide amide Affi-Gel 15 column. |
| Ultraviolet (UV) Absorbance |
| None |
| None |
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