| Haiqin Lu, Jie Zhu, Yuhui Zang, Yuguan Ze, Junchuan Qin
(2006)
Protein Expression and Purification,
46,
92-99 |
| Functional Studies |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| Rosetta-gami (DE3)pLysS E |
| 28.0 |
| 6 |
| pMD18-T |
| Bacteria were grown in 20 ml LB medium containing ampicillin 100 ug/ml, kanamycin 15 ug/ml, tetracycline 12.5 ug/ml, and chloramphenicol 34 ug/ml in a shaker incubator at 37 C until the OD600 nm reached 1.0 at which time 10 ml of the culture was added to 1000 ml 2YT medium containing ampicillin 100 ug/ml, kanamycin 15 ug/ml, tetracycline 12.5 ug/ml, and chloramphenicol 34 ug/ml and continue grown in a 37 C shaker for approximately 4 h until the OD600 nm reached 0.6-0.8 at which time the culture was grown in a shaker incubator at 28 C for about half an hour. Then, the culture was induced by 1 mM IPTG. After an incubation time of 6 h at 28 C, the cells were harvested by centrifugation. In a typical preparation, about 12 g of E. coli paste could be obtained from one liter of culture. |
| IPTG |
| OD 0.6-0.8 =
n/a |
| Sonication |
| None |
| None |
| not stated |
| Dilution into detergent |
| (20 mM Tris-HCl, 0.01 M NaCl, 0.1% Triton X-100 (v/v), and 2 mM CaCl2, pH 8.0 containing 2 M urea and once with the same buffer without urea |
| 20 mM Tris-HCl, 0.01 M NaCl, 2 mM CaCl2, and 20 mM DTT, pH 8.0) containing 2% Triton X-100, 4% Triton X-100, 6% Triton X-100, 8% Triton X-100, and 10 |
| 20 mM Tris-HCl, 0.01 M NaCl, 2 mM CaCl2, and 20 mM DTT, pH 8.0, 4% Triton X-100 |
| None |
| no tag |
| 8.0 |
| 4.0 |
| 2 |
| Overnight |
| DTT |
| 20 mM,20 mM,20 mM |
| The inclusion body-containing precipitate was washed three times with 50 ml buffer A (20 mM Tris-HCl, 0.01 M NaCl, 0.1% Triton X-100 (v/v), and 2 mM CaCl2, pH 8.0) containing 2 M urea and once with the same buffer without urea to remove the contaminants, and recovered by centrifugation. The recovered inclusion bodies were solubilized by serial extraction with buffer B (20 mM Tris-HCl, 0.01 M NaCl, 2 mM CaCl2, and 20 mM DTT, pH 8.0) containing 2% Triton X-100, 4% Triton X-100, 6% Triton X-100, 8% Triton X-100, and 10% Triton X-100 until most of the inclusion bodies were dissolved. In a typical preparation, most of the rhPON3 solubilized at 4% Triton X-100.
The obtained extraction solutions containing solubilized inclusion bodies were mixed and the protein concentration in the resulting solution was determined using BCA assay with micro BCA protein assay reagent. The protein concentration was adjusted to 2 mg/ml and the concentration of Triton X-100 was adjusted to 4%. The refolding was carried out under the air condition by stirring slowly overnight at 4 C.
|
| 8-Anilo-1-Naphthalenesulfonic Acid Fluorescence |
| None |
| Triton X-100 |
| 4% |
| 80mg prot/110mg |
| 90% |
| n/a |