| 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 to the culture. After 6 h 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,0.5 / 1.0 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 used for SDF-1a and CX3C chemokine fractalkine is same. |