| Ferreon JC, Ferreon AC, Li K, Lemon SM.
(2005)
J Biol Chem,
280,
20483-92 |
| Diagnostic studies |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| None |
| 37.0 |
| 4-5 h |
| pET21d |
| Protein Overexpression and Purification—TRIF was cloned as a His tag fusion protein into pET21d vector (Novagen) and recovered from Escherichia coli grown in a 20-liter fermenter as follows. A single colony of E. coli, carrying the expression plasmid, was inoculated into a small flask (37 °C, overnight) and then transferred into 20 liters of LB broth supplemented with 100 µg/ml ampicillin. Cells were grown at 37 °C until the A600 reached 0.6–0.8, then induced by addition of 1 mM isopropyl 1-thio--D-galactopyranoside followed by further incubation at 37 °C for 4–5 h. Cells were harvested and stored at –80 °C prior to purification. |
| IPTG |
| OD 0.6-0.8 =
600 |
| Not stated |
| None |
| Ion-exchange chromatography |
| insoluble |
| Dialysis |
| M urea, 100 mM sodium phosphate, 10 mM Tris, 2 mM -mercaptoethanol, pH 7.5) containing 50 mM imidazole |
| 6 M guanidine HCl, 100 mM sodium phosphate, 10 mM Tris, 2 mM -mercaptoethanol, pH 8, 5 ml of buffer/g of cell pellet |
| 20 mM Tris, 2 mM DTT, pH 7.5 |
| Ion-exchange chromatography |
| yes |
| 7.5 |
| 25.0 |
| n/a |
| n/a |
| DTT |
| 2 mM |
| Immunoblots suggested that almost all of the expressed TRIF was present in inclusion bodies; therefore purification was carried under denaturing conditions. The cell pellet was dissolved in extraction buffer (6 M guanidine HCl, 100 mM sodium phosphate, 10 mM Tris, 2 mM -mercaptoethanol, pH 8, 5 ml of buffer/g of cell pellet) for 24 h at 4 °C, with stirring. Cell debris was removed by ultracentrifugation, and the supernatant passed through a Ni2+ affinity fast protein liquid chromatography column. The column was rinsed with 10–15 volumes of wash buffer (8 M urea, 100 mM sodium phosphate, 10 mM Tris, 2 mM -mercaptoethanol, pH 7.5) containing 50 mM imidazole. Bound proteins were eluted with wash buffer containing 500 mM imidazole. The initial fractions contained TRIF and other low molecular mass contaminants, whereas the final fractions contained mostly TRIF (>90% purity). Fractions with impurities were subjected to size exclusion chromatography (Superdex 200). TRIF was refolded by dialysis against 20 mM Tris, 2 mM DTT, pH 7.5, with the signature of the folded protein monitored by fluorescence and CD spectroscopy. From a 100-liter large scale purification, 50 mg was obtained with purity of >90%. The majority of the contaminants appeared to be TRIF fragments or degradation products. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UTMB Protein Facility) confirmed that the molecular mass was 77.2 kDa. |
| Circular Dichroism (uv-CD) |
| None |
| None |
| n/a |
| n/a |
| >90% |
| n/a |