| Haidong Tan, Jinxia Wang, Zongbao (Kent) Zhao
(2007)
Protein Expression and Purification,
1,
1 |
| Recombinant Protein Expression |
| N-terminal GST |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 30.0 |
| 12 |
| pET41a |
| Escherichia coli BL21 (DE3) was transformed by plasmid pET41a-EK by electroporation and grown on LB agar plates supplemented with 30 ?g/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 ?g/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 600 =
0.1 |
| Sonication |
| None |
| Affinity chromotography |
| partial |
| Dialysis |
| 2 M urea, 0.25% ?-mercaptoethanol (V/V) and 1% Triton X-100 (V/V) |
| 8 M urea, 1% ?-mercaptoethanol (V/V), 0.1% DOC (W/V) and 1% Sarkosyl (W/V) |
| 500 mM NaCl, 20 mM Tris–HCl, pH 8.0 supplemented with 20% glycerol |
| Affinity chromotography |
| yes |
| 8.0 |
| 4.0 |
| n/a |
| 3 x 4h |
| None |
| n/a,n/a,n/a |
| The 2 M-urea-resistant insoluble portion was further treated with 25 mL of the solution containing 8 M urea, 1% ?-mercaptoethanol (V/V), 0.1% DOC (W/V) and 1% Sarkosyl (W/V). 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 |
| Glycerol |
| 20% |
| 150mg |
| 95% |
| n/a |