Refolding Record:
| Protein | |
|---|---|
| Protein Name | Human Granulocyte colony-stimulating factor |
| Abbreviated Name | hG_CSF |
| SCOP Family | Long-Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P09919 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Unknown |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 177 |
| Molecular Weight | 18987.0 |
| Pi | 5.42 |
| Molecular Weight | 18987.0 |
| Disulphides | 2 |
| Full Sequence |
TPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wang C, Wang L, Geng X. (2008) Biotechnol Prog, 24, 209-213 |
| Project Aim | Protein refolding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | DH5a |
| Expression Temp | 30.0 |
| Expression Time | 4 h |
| Expression Vector | pBV220 |
| Expression Protocol | Expression of rhG-CSF and Solubilization of rhG-CSF Inclusion Bodies. rhG-CSF was expressed in E. coli in the form of inclusion bodies, then the inclusion bodies were recovered by washing and centrifugation and finally solubilized in 8.0 mol·L-1 of urea, 0.05 mol·L-1 of Tris, 0.1 mol·L-1 of -mercaptolethanol, and 1 mmol·L-1 of EDTA. All the detailed procedures are the same as those demonstrated in the previous work (Wang et al., 2006a). Protein concentration in the supernatant was measured to be 2.3 mg·mL-1 according to the Bradford method. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 600 |
| Cell Disruption Method | Freeze/Thaw+Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Column refolding: Size-exclusion chromatography |
| Wash Buffer | 0.02 mol · L-1 Tris-HCl (pH 8.0) |
| Solubilization Buffer | 8.0 mol·L-1 of urea, 0.05 mol·L-1 of Tris, 0.1 mol·L-1 of -mercaptolethanol, and 1 mmol·L-1 of EDTA |
| Refolding Buffer | 0.1 mol/L of Tris, pH 8.0, 1.0 mmol/L of EDTA, 0.15 mol/L of NaCl, 15% glycerol (v/v), 2.5 mmol/L of GSH, 0.8 mmol/L of GSSG) to solution B (solution A containing 8.0 mol/L of urea) |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2.5/0.8 mM/L |
| Refolding Protocol | Refolding of rhG-CSF by Urea Gradient SEC. The hromatographic run was carried out at room temperature with use a SEC column (20 cm × 1.6 cm i.d.) packed with Superdex 75 gel. The column was first equilibrated with the mixed solution of expected ratio of solution A (0.1 mol/L of Tris, pH 8.0, 1.0 mmol/L of EDTA, 0.15 mol/L of NaCl, 15% glycerol (v/v), 2.5 mmol/L of GSH, 0.8 mmol/L of GSSG) to solution B (solution A containing 8.0 mol/L of urea) followed by a linear gradient to the urea concentration of 8.0 mol/L-1 (100% solution B). After equilibration in this manner, various volumes of denatured/reduced rhG-CSF prepared above were directly injected into the column and eluted with solution B at a flow rate of 2.0 mL/min. Detection was set at 280 nm. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | Glycerol |
| Additives Concentration | 15%(v/v) |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | n/a |