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 (2007) Biomedical chromatography, 1, in press |
| 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 | Details not given see following reference (Wang et al., 2006, J. Liq. Chrom. and Rel. Tech. vol. 29 p 203-217. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD 1.0 = 600 |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | column refolding: ion-exchange chromatography |
| Wash Buffer | 0.05 mol/L Tris, pH 8.0, 3.0 mol/L urea, 1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG |
| Solubilization Buffer | 8.0 mol/L urea solution containing 0.05 mol/L Tris (pH 8.0), 1 mmol/L EDTA and 0.1 mol/L β-mercaptoethanol |
| Refolding Buffer | 0.05 mol/L Tris, pH 8.0, 1.0 M NaCl, 3.0 mol/L urea, 1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 8.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 2.8 mg/ml |
| Refolding Time | n/a |
| Redox Agent | GSH/GSSG |
| Redox Agent Concentration | 2.5/0.8 mM,2.5/0.8 mM |
| Refolding Protocol | Procedures for the refolding and purification of rhG-CSF by IEC. Chromatographic runs were carried out at room temperature using a IEC column (20 × 1.2 cm i.d.) packed with Q Sepharose FF media with a bed volume of about 20 mL and connected to an ÄKTA Explorer 100A chromatographic system. The column was equilibrated with solution A consisting of 0.05 mol/L Tris, pH 8.0, 3.0 mol/L urea, 1.0 mmol/L EDTA, 2.5 mmol/L GSH and 0.8 mmol/L GSSG. The sample solution (400 μL) containing the solubilized and denatured rhG-CSF with 2.8 mg/mL total protein (containing 1.15 mg/mL of rhG-CSF) in 8.0 mol/L urea solution was directly injected into the column. After washing the column with 30 mL of solution A, the refolding with simultaneous purification of rhG-CSF was accomplished after a linear gradient elution from 100% A to 60% B (solution B consisted of solution A plus 1.0 mol/L NaCl) in 90 mL at a flow rate of 5.0 mL/min with detection at 280 nm. The column was regenerated using 20 mL of 100% B. |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | n/a |
| Refolding Yield | n/a |
| Purity | n/a |
| Notes | NOTE - protein was refolded into a buffer gradient from 0 to 60% buffer B. See above protocol. |