| Siemeister G, Schnurr B, Mohrs K, Schachtele C, Marme D, Martiny-Baron G
(1996)
Biochemical and Biophysical Research Com,
222,
249-255 |
| Drug Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3h |
| pET3d |
| Cells were grown in 250ml LB medium containing 100microg/ml ampicillin at 37degC. When OD600 reached 0.8, IPTG was added to a final concentration of 0.4mM and the culture was grown for another 3h. Cells were harvested by centrifugation (10min, 5000rpm, 4degC) and the pellet was resuspended in 2-ml of 20mM TrisHCl, 150mM NaCl pH 7.5. |
| IPTG |
| OD 600 =
0.8 |
| Sonication |
| Lysozyme |
| Ion-exchange + size exclusion chromatography |
| insoluble |
| Dilution |
| 50mM TrisHCl, 10mM 2-mercaptoethanol, 2mM EDTA, 5%(v/v) glycerol, 0.2mg/ml lysozyme, 10microg/ml DNaseI, 0.05% Na-deoxycholate, 1% NP-40 pH 8.0 |
| 6M urea, 0.1M DTT, 50mM MES pH 5.5 |
| PBS, 50mM glycine |
| Ion-exchange + size exclusion chromatography |
| no tag |
| 7.4 |
| 4.0 |
|
|
| None |
| n/a |
| The cell suspension was centrifuged again (10min, 5000rpm, 4degC) and the pellet was then frozen at -80degC, thawed at 37degC and resuspdended in 25ml wash buffer without Na-deoxycholate or NP-40 and incubated for 30min at 22degC.
The suspension was sheared by five high-speed treatments of 20s in an Ultra-Turrax dispersing apparatus and incubated for 10min at 22degC. The mixture was cooled on ice and sonicated six times (15s). After addition of Na-deoxycholate (0.05%) and NP-40(1%(w/v)), the mixture was incubated for 10min at 4degC and then centrifuged (10000rpm, 4degC, 30min). The pellet was then resuspended in 25ml wash buffer and recentrifuged. The inclusion body pellet was then resuspended in 25ml solubilization buffer at 4degC.
The solubilized protein was applied to a MonoS cation exchange column equilibrated with solubilization buffer. The protein was eluted between 150 and 200mM NaCl. Selected fractions were then passed down a Superdex 200 column equilibrated in solubilization buffer. Peak fractions were then pooled and mixed with two volumes of 6M urea, 0.5M cystamine, 0.1M glycine, 20mM Hepes pH 7.4 and incubated for 4h at 4degC with gentle agitation.
The protein was then refolded by sequential dilution to a final concentration of 0.3M urea by the addition of PBS/50mM Glycine. The refefolded protein was then purified by Ni2+ chelating affinity chromatography on a 1mL Ni-NTA resin, followed by buffer exchange by ultrafiltration to 0.1 N acetic acid
|
| SDS-PAGE |
| None |
| None,Glycine |
| 50mM |
| 5-10mg/L culture |
| 95% |
| Similar procedure also for VEGF121 reported in the same paper |