| Hao Y, Huang X, Mei X, Li R, Zhai Z, Yin S, Huang Y, Luo Y.
(2008)
Protein Expression and Purification,
60,
221-4 |
| Over expression & Renaturation |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 4 h |
| pET30a(+) |
| A single-colony transformant was inoculated into LB medium containing 50 μg/mL kanamycin and grown at 37 °C. The overnight culture was 50-fold diluted to inoculate in 100 mL fresh LB medium. When the optical density (OD600) reached about 0.6, IPTG was then added to a final concentration of 1 mM to induce expression of fusion protein for 4 h at 37 °C [11]. Cell pellets harvested were suspended in 1 mL PBS buffer (pH 7.4) and were subjected to sonication in ice bath at 300 W with 10 s on/off cycle to prevent overheating, for a total time of 6 min. The total cellular proteins were then partitioned into soluble and insoluble fractions by centrifugation at 12,000g for 10 min at 4 °C. In order to determine the solubility of fusion protein, the samples were analyzed by SDS–PAGE.
|
| IPTG |
| OD 0.6 =
600 |
| Sonication |
| None |
| Metal affinity chromatography |
| partial |
| Dilution/Dialysis combination |
| 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 0.5% (v/v) Triton X-100 |
| 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 20 mM β-ME and 8.0 M urea (pH 7.4). |
| PBS buffer containing 1 mM EDTA, 1% glycine, 5% glycerol, 0.9 mM GSH, 0.1 mM GSSG |
| Metal affinity chromatography |
| no |
| 0.0 |
| 4.0 |
| n/a |
| n/a |
| GSH/GSSG |
| 0.9/0.1 mM |
| The inclusion bodies in insoluble fraction were washed twice with buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl and 0.5% (v/v) Triton X-100. The washed inclusion bodies were further dissolved in binding buffer containing 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 20 mM β-ME and 8.0 M urea (pH 7.4). After solubilization, the recombinant protein was purified by affinity chromatography. Ni sepharose 6 Fast Flow media was packed and equilibrated with binding buffer. The target protein was eluted with a stepwise gradient of imidazole from 20 to 200 mM. Purified proteins were refolded by urea gradient dialysis at 4 °C in PBS buffer containing 1 mM EDTA, 1% glycine, 5% glycerol, 0.9 mM GSH, 0.1 mM GSSG. Meanwhile, urea was added into the PBS buffer every 12 h at a gradient concentration (6, 4, 2, 1, 0.5 and 0 M). |
| Activity assay |
| None |
| Glycerol,Glycine |
| 5%/1% |
| n/a |
| n/a |
| n/a |