| Le Moigne V, Robreau G, Borot C, Guesdon JL, Mahana W.
(2005)
Tuberculosis (Edinb).,
85,
213-219 |
| Identification and Characterization |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| BL21(DE3) |
| 37.0 |
| 3-4 h |
| pET15b |
| The PCR products were digested by the two restriction enzymes NdeI and XhoI and ligated (16 h at 4 °C with 1 U T4 ligase; Eurogentec, Belgium) into the NdeI/XhoI-digested pET15b plasmid. E. coli DH5 (Invitrogen, France) was transformed with the plasmid and transformants were picked and tested for the presence of the insert. Positive transformants were sequenced (Genomexpress, France). The plasmid with the insert was used to transform E. coli strains BL21 (DE3) (Novagen, France) for overexpression of the protein.
Bacteria from overnight culture were diluted (1:10) in fresh 2×TY media and grown to mid log-phase (OD600=0.6), then 1 mM of isopropyl-β-d-thiogalactopyranoside (IPTG) was added and the cultures were harvested after 3–4 h of incubation. |
| IPTG |
| OD 0.6 =
600 |
| None |
| None |
| Ni-NTA column |
| insoluble |
| Dialysis |
| 100 mM Na2HPO4, 10 mM Tris-HCl, 8 M urea, pH 8.0 |
| urea 8 M, first at pH 5.9 then at pH 4.5 |
| phosphate-buffered saline (PBS) pH 7.4 |
| Ni-NTA column |
| no tag |
| 7.4 |
| 4.0 |
| n/a |
| n/a |
| None |
| n/a |
| Bacterial cell pellets were collected by centrifugation and resuspended in lysis buffer (100 mM Na2HPO4, 10 mM Tris-HCl, 8 M urea, pH 8.0). After centrifugation (10 000g, 30 min) the supernatant was removed and incubated for 1 h at room temperature with Ni-NTA resin (Qiagen, France). After two washes with the lysis buffer pH 6.3, the recombinant protein was eluted with urea 8 M, first at pH 5.9 then at pH 4.5. The purified recombinant proteins were renatured by dialysis against phosphate-buffered saline (PBS) pH 7.4 at 4 °C and finally filtered on a 0.2 μm filter and stored in the same buffer. |
| Western Blot,SDS-PAGE,ELISA |
| None |
| None |
| n/a |
| n/a |
| n/a |
| n/a |