| Schmid B, Kromer M, Schulz GE
(1996)
FEBS Letters,
381,
111-114 |
| Crystallography |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3)pLysS |
| 37.0 |
| 6h |
| pET-3b |
| 250ml SB medium (20g/L caein hydrolysate, 10g/L yeast extract, 5g/L NaCl, 2.5g/L K2HPO4, 1g/L MgSO4.7H2O, pH 7.0) containing 100mg/L ampicillin and 50mg/L chloramphenicol was inoculated with 10mL starter culture previously incubated for 8hr at 37degC. Incubation of the larger culture continued at 37degC with shaking until OD578 reached 0.8. Protein expression was induced with 1mM IPTG and the cultures were incubated a further 6h. Cells were harvested by centrifugation (10800xg, 15min) then resuspended in 3ml of TEN buffer (50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl) per gram of cells (wet weight) and frozen overnight. |
| IPTG |
| OD 578 =
0.8 |
| Sonication |
| None |
| None |
| insoluble |
| Dilution/Column Refolding Combination |
| 2% Triton X-100, 50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl |
| 8M Urea, 50mM TrisHCl pH 8.0, 10mM EDTA, 100mM NaCl |
| 0.6% (w/v) n-octyltetraoxyethylene (C8E4), 20mM TrisHCl pH 7.2, 100mM LiCl, 3mM NaN3 |
| None |
| no tag |
| 7.2 |
| 25.0 |
|
|
| None |
| n/a,n/a |
| Following thawing, benzonase (50U) was added and the suspension stirred at room temperature for 30min. Cells were sonicated for 20min and the inclusion bodies collected by centrifugation (4300xg, 20min). The pellet was resuspended in 60ml wash buffer and incubated overnight at 37degC with shaking. The inclusion bodies were then centrifuged again and resuspended in 60ml of TEN buffer (2hr shaking at 37degC) followed by a final round of centrifugation, yielding a white pellet of inclusion bodies.
The inclusion bodies were then resuspended in solubilization buffer, incubated at 37deg for 2h with mild shaking and centrifuged (48000xg, 20min). The supernatant volume was increased to 125ml with solubilization buffer and then further diluted with 125ml of LDAO-10 buffer (10%(w/v) N,N-dimethyldodecyl-amine-N-oxide (LDAO) in TEN buffer) and sonicated for 30min. The solution was then loaded onto a Q-sepaharose column which had been equilibrated with LDAO-0.2 buffer (0.2%(w/v) LDAO, 50mM TrisHCl pH 8.0, 100mM NaCl, 3mM NaN3). After washing with LDAO-0.2 buffer, the column was washed further with C8E4 buffer (0.6% (w/v) n-octyltetraoxyethylene (C8E4), 20mM TrisHCl pH 7.2, 100mM LiCl, 3mM NaN3). The protein was then eluted using a 100-600mM LiCl gradient in C8E4 buffer. 2ml fractions were collected and suitably pure fractions pooled and dialyzed against C8E4 buffer containing 300mM LiCl. |
| Crystallography |
| None |
| None |
|
| 20-25mg/ml |
|
|