| Tan H, Wang J, Zhao ZK.
(2007)
Protein Expression and Purification,
56,
40-47 |
| Protein refolding |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 48 h |
| pET41a |
| Escherichia coli BL21 (DE3) was transformed by plasmid pET41a-EK by electroporation and grown on LB agar plates supplemented with 30 μg/mL kanamycin for 24 h. One colony from the plate was transferred into 6 mL LB medium, incubated at 37 °C, 200 rpm for 12 h. The pre-culture was diluted 100-fold to inoculate 500 mL into LB medium containing 30 μg/mL kanamycin. The culture was allowed to grow with shaking (200 rpm) at 37 °C, induced with 0.1 mM IPTG and 2% glucose when OD600 reached 0.1, and incubated at 30 °C for an additional 12 h. |
| IPTG + glucose |
| OD 0.1 =
600 |
| Sonication |
| None |
| Ni-NTA agrose chromatography |
| partial |
| Dilution |
| 2 M urea, 0.25% β-mercaptoethanol (V/V) and 1% Triton X-100 (V/V) |
| 8 M urea, 1% β-mercaptoethanol (V/V), 0.1% DOC (W/V) and 1% Sarkosyl (W/V) |
| 500 mM NaCl, 20 mM Tris–HCl, pH 8.0 |
| Ni-NTA agrose chromatography |
| no |
| 8.0 |
| 25.0 |
| n/a |
| n/a |
| None |
| n/a |
| The culture broth was centrifuged at 8000 rpm for 10 min to yield 3.0 g of wet cells. Cells were resuspended in 30 mL of buffer containing 20 mM Tris–HCl, 500 mM NaCl, pH 8.0 and lysed by sonication. The cell extracts were centrifuged at 8000g for 15 min and the supernatant was recovered (Fig. 2I). The precipitate was washed twice with 20 mL of solution containing 2 M urea, 0.25% β-mercaptoethanol (V/V) and 1% Triton X-100 (V/V). The supernatants and the washes (Fig. 2II) were further purified by Ni–NTA affinity chromatography. The 2 M-urea-resistant insoluble portion was further treated with 25 mL of the solution containing 8 M urea, 1% β-mercaptoethanol (V/V), 0.1% DOC (W/V) and 1% Sarkosyl (W/V) (Fig. 2III). Formulation of this media is based on the strong hydrophobic interaction of DOC with protein [16] and the excellent aggregates rupture ability of Sarkosyl [17].
The resulting suspension was centrifuged at 8000g for 20 min and the debris was discarded (Fig. 2IV). The supernatant (25 mL) was collected and diluted 5-fold with ice cold buffer A (500 mM NaCl, 20 mM Tris–HCl, pH 8.0) supplemented with 20% glycerol, and dialyzed with 1 L of buffer A using three buffer changes (4 h for each change). |
| Enzyme activity |
| None |
| None |
| n/a |
| 40% |
| n/a |
| n/a |