Refolding Record:
| Protein | |
|---|---|
| Protein Name | Granulocyte colony-stimulating factor |
| Abbreviated Name | G-CSF |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P09919 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Monomer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 179 |
| Molecular Weight | 19058.1 |
| Pi | 5.43 |
| Molecular Weight | 19058.1 |
| Disulphides | Unknown |
| Full Sequence |
ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Ishibashi M, Tokunaga H, Arakawa T, Tokunaga M. (2001) Protein Expression and Purification, 21, 317-322 |
| Project Aim | Over expression & Renaturation |
| Fusion | N-terminal hexahistidine tag |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21(DE3) |
| Expression Temp | 37.0 |
| Expression Time | 3 h |
| Expression Vector | pETIgSE |
| Expression Protocol | Plasmid pETIgSE was used to transform E. coli BL21(DE3) containing a LacUV5-promoter-driven T7 RNA polymerase. The transformant grew normally. BL21(DE3) harboring pETIgSE was grown in 125 ml of LB–ampicillin at 37C for 3 h and synthesis of the His-Ig domain was induced by the addition of 0.2 mM Purified His-Ig protein isopropyl-1-thio-􏰍-D-galactopyranoside. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD n/a = n/a |
| Cell Disruption Method | Ultrasonic oscillator |
| Lytic Agent | None |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | n/a |
| Solubilization Buffer | 2.5 ml of 100 mM Na-phosphate/10 mM Tris buffer (P buffer), pH 7.4, containing 8 M urea |
| Refolding Buffer | 6 M urea, 25 mM Tris –HCl buffer, pH 8.0, 5 mM dithiothreitol |
| Pre-Refolding Purification | Ni-NTA agrose chromatography |
| Tag Cleaved | no |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | DTT |
| Redox Agent Concentration | 5 mM,5 mM |
| Refolding Protocol | After 2-h induction of His-Ig protein synthesis, cells were harvested and disrupted by sonic oscillation (Branson sonifier with 1/2 in. tip) for 3 min with 50% pulse in 2 ml of 50 mM Tris –HCl buffer, pH 7.5. The crude homogenate was centrifuged at 12,000g for 20 min. The pellet fraction was dissolved in 2.5 ml of 100 mM Na-phosphate/10 mM Tris buffer (P buffer), pH 7.4, containing 8 M urea, sonicated briefly, and incubated at 37􏰊C for 30 min with gentle shaking to solubilize proteins efficiently. Dissolved proteins in supernatant after centrifugation at 12,000g for 20 min were applied to an Ni-NTA column (QIAGEN, 0.5 ml resin in Bio-Rad PolyPrep column No. 731-1550) equilibrated with the P buffer, and eluted in a stepwise manner with 1.2 ml of P buffer containing 8 M urea, at a pH adjusted to 7.4, 6.6, 5.5, and 4.0 (three times each). To refold the His-Ig protein, the material eluted at a pH below 5.5 was diluted to a protein concentration of 􏰋90 􏰎g/ml with 6 M urea, 25 mM Tris –HCl buffer, pH 8.0, and then dialyzed against 500 ml of the same buffer containing 5 mM dithiothreitol at 4􏰊C overnight, with a change of buffer. Urea was removed from this sample by dialysis against 500 ml of 10 mM Tris –HCl, pH 8.0, 1 mM EDTA, 2 mM reduced form and 0.01 mM oxidized form of glutathion at 4c for 4 days. The dialysis buffer was changed once during this 4-day refolding proces |
| Refolding Assay | Binding assay |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | 4.8% |
| Purity | 12 mg/L |
| Notes | n/a |