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 | 178 |
| Molecular Weight | 19058.1 |
| Pi | 5.43 |
| Molecular Weight | 19058.1 |
| Disulphides | 2 |
| Full Sequence |
ATPLGPASSLPQSFLLKCLEQVRKIQGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHSGLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPAFASAFQRRAGGVLVASHLQSFLEVSYRVLRHLAQP
|
| 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 | Dilution |
| Wash Buffer | 0.02 mol · L-1 Tris-HCl (pH 8.0) |
| Solubilization Buffer | 8.0 mol/L of urea, 0.05 mol/L of Tris, 0.1 mol/L of B-mercaptolethanol, and 1 mmol/L of EDTA. |
| Refolding Buffer | 3.0 mol/L of urea, 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, and 0.8 mmol/L of GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | n/a |
| Refolding Time | 24 h |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2.5/0.8 mM/L |
| Refolding Protocol | Refolding of rhG-CSF by Dilution Method. Various volume of denatured/reduced rhG-CSF prepared above were diluted with a refolding buffer containing 3.0 mol·L-1 of urea, 0.1 mol·L-1 of Tris, pH 8.0, 1.0 mmol·L-1 of EDTA, 0.15 mol·L-1 of NaCl, 15% glycerol (v/v), 2.5 mmol·L-1 of GSH, and 0.8 mmol·L-1 of GSSG to a volume of 15 mL, which corresponds to the width of rhG-CSF in the urea gradient SEC, and the solution was incubated at room temperature for 24 h. |
| 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 |