| Veldkamp CT, Peterson FC, Hayes PL, Mattmiller JE, Haugner JC 3rd, de la Cruz N, Volkman BF.
(2007)
Protein Expression and Purification,
52,
202-209 |
| NMR |
| N-terminal hexahistidine tag |
| Protein recombinantly expressed as and refolded from inclusion bodies. |
| Escherichia coli |
| SG13009 [pREP4] |
| 37.0 |
| 6 h |
| pQE30 |
| Escherichia coli strain SG13009[pREP4] containing the SDF-1α expression plasmid was grown at 37 °C in either 1 L of Luria-Bertani or M9 minimal media containing 15NH4Cl as the sole nitrogen source. Once an OD600 of 0.7 was reached expression was induced through the addition of 1mM IPTG. After 6 h the cells were harvested by centrifugation at 5000g and stored at −80 °C. |
| IPTG |
| OD 600 =
0.7 |
| French Press |
| None |
| None |
| insoluble |
| Column refolding: Nickel-chelating chromatography |
| 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, and 0.1% (v/v) 2-mercaptoethanol |
| 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, and 6 M guanidinium hydrochloride |
| 20 mM Tris pH 8.0, 100 mM NaCl, 5 mM β-cyclodextrin, 1 mM reduced glutathione, 0.5 mM oxidized glutathione |
| None |
| yes |
| 8.0 |
| 25.0 |
| n/a |
| 1 h |
| GSH/GSSG |
| n/a,1 / 0.5 mM |
| Protein purification and on-column refolding
The procedures for solution and on-column refolding of SDF-1α are the same up to the point of batch loading the Ni sepharose™ 6 column and the 30 min incubation. After incubation, the column was washed with 100 mL of detergent buffer (20 mM Tris pH 8.0, 100 mM NaCl, 1% Triton X-100 (v/v), 10 mM 2-mercaptoethanol) followed by 100 mL of oxidation buffer (20 mM Tris pH 8.0, 100 mM NaCl, 5 mM β-cyclodextrin, 1 mM reduced glutathione, 0.5 mM oxidized glutathione) and 70 mL of wash buffer (20 mM Tris pH 8.0, 500 mM NaCl). The refolded SDF-1α containing correctly oxidized disulfide bonds was eluted with elution buffer (25 mM Hepes pH 7.4, 300 mM NaCl, 1 M imidazole). Fractions containing SDF-1α were pooled and dialyzed against 4 L of 0.3% acetic acid and then lyophilized for CNBr cleavage. MALDI-TOF MS analysis of the SDF-1α was done before and after the CNBr cleavage to check for hexahistadine tag removal. After the CNBr cleavage the refolded, native N-terminal SDF-1α was lyophilized and purified to >98% homogeneity by RP HPLC. On-column refolding and purification of RANTES/CCL5, lymphotactin/XCL1, and fractalkine/CX3CL1 were performed in a similar fashion. |
| Unspecified |
| None |
| None,β-cyclodextrin |
| 5mM |
| n/a |
| n/a |
| The refolding/purification protocol for SDF-1a and CC Chemokine Lymphotactin is same |