| Fernández MM, De Marzi MC, Berguer P, Burzyn D, Langley RJ, Piazzon I, Mariuzza RA, Malchiodi EL.
(2006)
Molecular Immunology,
43,
927-38 |
| Undefined |
| None |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| B834(DE3) |
| 0.0 |
| 00 |
| n/a |
| HLA-DR1 was produced as described by Frayser et al. (1999). Briefly, plasmids encoding the HLA-DR1 chain (DR*0101) and β chain were transformed separately into E. coli BL21 (DE3) cells. Expression was induced by addition of 1 mM of IPTG at an OD600 of 0.6–0.8. Inclusion bodies (IB) were extensively washed and the subunits purified under denaturing and reducing conditions using anion exchange chromatography on a HQ50 column (Perseptive Biosystems, Cambridge, MA). Yield was 16 and 20 mg/l growth medium for and β subunit, respectively. |
| IPTG |
| OD 0.6-0.8 =
600 |
| Not stated |
| None |
| Ion-exchange chromatography |
| insoluble |
| Dilution |
| n/a |
| n/a |
| 20 mM Tris–HCl, 25% (w/v) glycerol, 0.5 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidized glutathione, pH 8.5 |
| Ion-exchange chromatography |
| no tag |
| 8.5 |
| 4.0 |
| n/a |
| 2 days |
| GSH/GSSG |
| 3/0.3 mM |
| Purified subunits were diluted dropwise with constant stirring to a final concentration of 50 μg/ml into a refolding solution of 20 mM Tris–HCl, 25% (w/v) glycerol, 0.5 mM EDTA, 3 mM reduced glutathione and 0.3 mM oxidized glutathione, pH 8.5 and kept at 4 °C for 2 days in the presence of 1 μM HA-peptide, residues 306–318 (PKYVKQNTLKLAT), from influenza hemaglutinin. Recombinant HLA-DR1 was purified from the folding mixture using a Mono Q column, from which it eluted as a major peak at 15% of buffer B (20 mM Tris–HCl pH 8, 1 M NaCl). |
| Unspecified |
| None |
| Glycerol |
| 25% |
| n/a |
| n/a |
| n/a |