| Li D., Yu J.-F., Chen Y.-J., Ma H.-B., Wang Z.-F., Zhu Y.-B., Zhang X.-G.
(2004)
Acta Biochemica et Biophysica Sinica,
36,
141-146 |
| Functional Studies |
| N-terminal GST |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21 |
| 37.0 |
| 5h |
| pGEX-5x-3 |
| Cells were grown in 2ml culture medium containing 1% glucose, 50mg/ml ampicillin and 34mg/ml chloramphenicol overnight at 37degC. 500mL of LB media was then inoculated with the overnight culture and grown at 37degC until OD600 was 1.0. Protein expression was induced with 1mM IPTG and cells were grown for a further 5h. |
| IPTG |
| OD 600 =
1.0 |
| Chemical |
| None |
| None |
| not stated |
| Dilution |
| n/a |
| 8M Urea, 50mM TrisHCl pH 8.0, 100 mM NaCl, 2mM EDTA, 2mM DTT, 1mM PMSF |
| 2M Urea, 50mM TrisHCl pH 8.0, 100mM NaCl, 1% Tween20, 1mM PMSF, 1mM reduced glutathione, 0.2mM oxidized glutathione |
| None |
| no |
| 7.3 |
| 25.0 |
|
|
| GSH/GSSG |
| 1mM/0.2mM |
| Following centrifugation, the cell pellet was resuspended in 50ml solubilization buffer, and the suspension stirred slowly overnight at 4degC. The solution was centrifuged at 10000g for 30min and the insoluble pellet discarded. The supernatant was slowly dropped into 450ml refolding buffer then vigorously stirred. The refolding mixutre was incubated at 4degC for 3 days and then centrifuged to remove insoluble materials. The supernatant was then dialyzed against 5L Buffer A (140 mM NaCl, 2.7mM KCl, 10 mM Na2HPO4, 1.8mM KH2PO4 pH 7.3) for 18h at 4degC. Following centrifugation, 250ml of the supernatant was filtered and loaded onto a 1ml GSTrapFF affinity chromatography column equilibrated with Buffer A. Bound proteins were eluted with 50mM TrisHCl pH 8.0 with 10mM glutathione. Eluted fractions were collected and loaded onto a Bio-Gel P-100 column equilibrated in 50mM TrisHCl pH 8.0, 250mM NaCl. The pooled fractions containing GST-PD-1 were collected and filtered. |
| Bioactivity |
| None |
| None |
|
| 13mg per L |
| 95% |
|