| Klein H, Löschner B, Zyto N, Pörtner M, Montag T.
(1998)
Glycoconj J.,
15,
147-153 |
| Purification & characterization |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| M15 |
| 37.0 |
| 5 h |
| pQE-30 |
| E. coli cells transformed with pQE30-rSML were grown overnight with shaking at 37 °C in 2;YT medium (20 ml) containing ampicillin (100 ug/ ml ). The inoculant culture was added to growth medium (1 litre 2;YT supplemented with 100 ug/ ml ampicillin), incubated at 37 °C until optical density (OD) 590 had reached a value of 0.8, and then induced for 5 h with 2 mM isopropyl thiogalactoside (IPTG). Bacteria were harvested by centrifugation (4000;g, 20 min), and lysed in 8 M urea, 10 mM Tris/HCl, pH 8.0, 100 mM Na-phosphate at 5 ml/ g wet weight for 1 h at room temperature. After centrifugation at 10 000;g for 30 min, the supernatant was collected and loaded, at a flow rate of 10 — 15 ml/h, onto a 4 ml Ni-NTA column (Qiagen, Germany) pre-equilibrated in lysis buffer. The resin was washed with 10 column volumes of lysis buffer until the OD 280 of the flow-through was below 0.01. After washing with the same buffer at pH 6.0 (OD 280 of the flow-through below 0.01) bound proteins were eluted with buffer containing 250 mM imidazole [14]. |
| IPTG |
| OD 0.8 =
590 |
| Not stated |
| None |
| Ni-NTA column |
| insoluble |
| Dilution |
| 8 M urea, 10 mM Tris/HCl, pH 8.0, 100 mM Na-phosphate |
| 8 M urea, 10 mM Tris/HCl, pH 8.0, 100 mM Na-phosphate |
| 10 mM Tris/HCl, 0.01% (v/v) Tween-80, 0.25 M NaCl, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), pH 8.5 |
| Ni-NTA column |
| no |
| 0.0 |
| 30.0 |
| n/a |
| 2 h |
| GSH/GSSG |
| 1/0.1 mM |
| Refolding of rSML (His)6 from E. coli Ni-NTA affinity purified rSML (His)6 was diluted in nine volumes (v/v) of folding buffer [10 mM Tris/HCl, 0.01% (v/v) Tween-80, 0.25 M NaCl, 1 mM reduced glutathione (GSH), 0.1 mM oxidized glutathione (GSSG), pH 8.5] to a final protein concentration of 50 ug/ ml by gently mixing at 30 °C. The total volume in the refolding assay was 20 ml. The solution was held at the same temperature for an additional two hours to allow refolding of the denaturated rSML(His)6 . After the refolding process, the pH was adjusted to 7.4. To remove residual denaturant, the suspension was dialysed extensively at 4 °C overnight against 200 — 300 volumes of the folding buffer. Subsequently, the protein solution was concentrated by vacuum ultrafiltration using collodion bags (Sartorius, Germany). |
| Immunoblot analysis |
| None |
| Tween 80 |
| 0.01% (v/v) |
| n/a |
| n/a |
| n/a |