Refolding Record:
| Protein | |
|---|---|
| Protein Name | Stromal cell-derived factor-1a |
| Abbreviated Name | SDF-1a |
| SCOP Family | interleukin 8-like chemokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P48061 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha+Beta |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 65 |
| Molecular Weight | 7610.0 |
| Pi | 9.69 |
| Molecular Weight | 7610.0 |
| Disulphides | 2 |
| Full Sequence |
VSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALN
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | 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 |
| Project Aim | NMR |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG13009 [pREP4] |
| Expression Temp | 37.0 |
| Expression Time | 6 h |
| Expression Vector | pQE30 |
| Expression Protocol | 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 isopropyl-β-d-thiogalatopyranoside (IPTG) to the culture at a final concentration of 1 mM. After 6 h cells were harvested by centrifugation at 5000g and stored at −80 °C. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 0.7 = 600 |
| Cell Disruption Method | French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Metal affinity chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, and 0.1% (v/v) 2-mercaptoethanol |
| Solubilization Buffer | 50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, and 6 M guanidinium hydrochloride |
| Refolding Buffer | 4 L of 0.3% (v/v) acetic acid. 2-mercaptoethanol |
| Pre-Refolding Purification | Metal affinity chromatography |
| Tag Cleaved | no |
| Refolding pH | 6.5 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | None |
| Redox Agent Concentration | n/a,n/a,n/a,n/a,n/a,n/a |
| Refolding Protocol | Protein purification and solution-based dialysis refolding Cell pellets were resuspended in 10 mL of buffer A (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride, and 0.1% (v/v) 2-mercaptoethanol). Cells were lysed by two to three passages through a French pressure cell, 16,000 psi. Inclusion bodies containing SDF-1α were isolated through centrifugation at 15,000g and the supernatant was discarded. The insoluble inclusion body pellet was solubilized using buffer AD (50 mM sodium phosphate pH 7.4, 300 mM NaCl, 10 mM imidazole, and 6 M guanidinium hydrochloride) and batch loaded onto 5 mL disposable columns containing 2 mL of Ni sepharose™ 6 fast flow resin (Amersham/GE Healthcare). After a 30 min incubation the column was washed 4× with 10 mL of buffer AD and eluted with buffer BD (50 mM sodium acetate pH 4.5, 300 mM NaCl, 10 mM imidazole, and 6 M guanidinium hydrochloride). The eluted SDF-1α was dialyzed twice against 4 L of 0.3% (v/v) acetic acid. 2-mercaptoethanol was added to the dialyzed SDF-1α to a final concentration of 0.1% (v/v). NaH2PO4 and NaCl to a final concentration of 50 mM were also added and the pH was raised to 6.5–6.75 with NaOH. Tobacco etch virus protease (TEV) was then added to this solution to remove the hexahistadine tag (1:1000 w/w). The disulfide bonds in SDF-1α were formed through diluting protease digestion solution from 30–40 to 150 mL with 20 mM Tris pH 8.0 to raise the pH followed by dialysis against 4 L of the same buffer. Dialysis-based SDF1-α oxidation fails if the hexahistadine tag is not removed before oxidation. After disulfide bond formation the SDF-1α was concentrated to 30 mL using ultrafiltration and acidified with HCl (pH < 3.0). SDF-1α was then purified to greater than 98% homogeneity using reverse phase (RP) HPLC in 0.1% aqueous trifluoroacetic acid (TFA) with a 30 min CH3CN gradient from 21 to 42%. Purified SDF-1α was lyophilyzed and then dissolved in 80% aqueous TFA with 100-fold molar excess of CNBr overnight. Removal of the residual N-terminal Gly-Met dipeptide (which remained after digestion with TEV protease) was verified by MALDI-TOF mass spectrometry. Cleaved SDF-1α was lyophilyzed and repurified by RP HPLC. Solution refolding and purification of RANTES [15], lymphotactin [16], and fractalkine [17] and [18] were performed as described elsewhere. |
| Refolding Assay | Unspecified,HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |