| Boesen CC, Motyka SA, Patamawenu A, Sun PD
(2000)
Protein Expression and Purification,
20,
98-104 |
| Undefined |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 30.0 |
| ? |
| pET15b |
| An overnight culture of cells grown in LB broth was added to 10L of Super Broth containing 50mg/L ampicillin (pET15b) or kanamycin (pET30a). Cells were grown at 37degC, 500rpm, pH 7.0 and 6.0psi O2 flow until OD600 reached 3.0-5.0. Expression was induced with 50mg/mL IPTG and the temperature lowered to 30degC until OD600 reached 10.0-15.0, at which time the cells were harvested. |
| IPTG |
| OD 600 =
3.0-5.0 |
| Microfluidizer |
| Lysozyme |
| None |
| insoluble |
| Dilution |
| 1.5M urea, 5mM DTT, 5mM EDTA, 0.25% Triton X-100, 0.1MTris pH 8.0 |
| 6M GdnHCl, 10mM DTT, 20mM Tris pH 7.0 |
| 0.5M arginine, 20mg/L AEBSF protease inhibitor, 2mM EDTA, 5mM cysteamine, 0.5mM cystamine, 0.1M Tris pH 8.0 |
| None |
| no tag |
| 8.0 |
| 4.0 |
|
| 16h |
| cysteamine/cystamine |
| 5mM/0.5mM,5mM/0.5mM |
| Cells were resuspended in 600ml of PBS containing 20mg/ml AEBSF protease inhibitor and lysed using a microfluidizer. Lysozyme and DNase I were added and the lysate was stirred overnight at 4degC. Following centrifugation (25000g, 40min, 4degC), the pellet was resuspended and washed four times with wash buffer, followed by two more cycles of centrifugation and resuspension with 3mM DTT in water. For refolding, 50mg of protein was dissolved in 10ml of solubilization buffer and then rapidly injected into 1L refolding buffer. The solution was stirred for 16h at 4degC and then dialyzed against water until the overall salt concentration was less than 10mM. After filtering, the refolding solution was loaded onto a SOURCE 15Q column and eluted in 25mM Tris pH 8.0 with a linear gradient fro 0 to 0.75M NaCl. The protein was then concentrated to 1.0ml, then loaded onto a MonoP column and eluted with a 0-0.5M NaCl gradient in 50mM Hepes pH 7.0. The protein was then concentrated again and passed through a Superdex75 column in 50mM NaCl, 50mM Tris pH 8.0. |
| SDS-PAGE |
| None |
| None,L-Arginine |
| 0.5M |
|
|
|