Lee EK, Hwang JH, Shin DY, Kim DI, Yoo YJ.
(2005)
Protein Expression and Purification,
40,
183-189 |
Over expression & Renaturation |
N-terminal hexahistidine tag |
Protein recombinantly expressed as and refolded from inclusion bodies. |
Escherichia coli |
BL21(DE3) |
37.0 |
3 h |
pET |
Transformed bacteria (BL21(DE3)/pET_H6Ub–Aβ42) were grown at 37 °C in Luria broth medium. When the absorbance reached 0.6 at 600 nm, protein expression was induced by adding 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG). After 3 h, cells were immediately chilled on ice and harvested by centrifugation at 5000g for 30 min at 4 °C. Cells were resuspended in buffer A (50 mM Tris, pH 8.0, 150 mM NaCl) containing 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride and ruptured by sonication on ice |
IPTG |
OD 0.5 =
600 |
Sonication |
None |
Ni-NTA column |
partial |
Dialysis |
buffer A (50 mM Tris, pH 8.0, 150 mM NaCl) containing 1 mM EDTA and 1 mM phenylmethanesulfonyl fluoride |
8 M urea, 20 mM Tris- HCl (pH 8.0) |
buffer A containing 50 mM imidazole |
Ni-NTA column |
no |
8.0 |
25.0 |
n/a |
n/a |
None |
n/a |
After centrifugation at 10,000g for 30 min, the pellet was washed twice with buffer A and then dissolved in buffer A containing 8 M urea. After centrifugation at 10,000g for 30 min, the supernatant was loaded directly onto a nickel–nitrilotriacetic acid (Ni–NTA) affinity column (Qiagen, Germany). The column was washed with 10 column volumes (CV) of buffer A containing 8 M urea followed by 10 CV of buffer A containing 50 mM imidazole. Bound proteins were eluted with buffer A containing 400 mM imidazole, and then concentrated by ultrafiltration and dialyzed in 10 mM Tris, pH 8.0. The protein concentration was determined using the Bradford method [21]. |
SDS-PAGE |
None |
None |
n/a |
4 mg/1 L |
25 mg/1 |
n/a |