Refolding Record:
| Protein | |
|---|---|
| Protein Name | granulocyte-macrophage colony-stimulating factor |
| Abbreviated Name | GM-CSF |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Mouse |
| UniProt Accession | P01587 |
| 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 | 126 |
| Molecular Weight | 14112.2 |
| Pi | 5.87041 |
| Molecular Weight | 14112.2 |
| Disulphides | 2 |
| Full Sequence |
APTRSPITVTRPWKHVEAIKEALNLLDDMPVTLNEEVEVVSNE
FSFKKLTCVQTRLKIFEQGLRGNFTKLKGALNMTASYYQTYCPPTPETDCETQVTTYADF
IDSLKTFLTDIPFECKKPGQK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Sebollela, A et al (2005) J Biol Chem, 280, 31949-56 |
| Project Aim | Structure-Function |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | BL21 |
| Expression Temp | 37.0 |
| Expression Time | 4 h |
| Expression Vector | pGEX-3x |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | Washing inclusion body |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | Tris 50 mM, pH 8.0 50 mM KCl |
| Solubilization Buffer | Tris 50 mM, pH 8.0 + 6 M urea |
| Refolding Buffer | phophate 20 mM + 150 mM NaCl pH 7.5 |
| Pre-Refolding Purification | Washing inclusion body |
| Tag Cleaved | yes |
| Refolding pH | 7.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | E. coli (strain BL21(DE3)pLysS) was transformed with pGEX-3XmGM-CSF and the expression of recombinant GST-fusion protein was induced by addition of 0.3 mM IPTG. After 4 h at 37 oC, the cells were washed and disrupted, and proteins were recovered from inclusion bodies after solubilization with 6 M urea. Urea was removed by dialysis against 20 mM potassium phosphate, 150 mM NaCl, pH 7.5. Refolded fusion proteins were purified by affinity chromatography using a GSTrap 1-mL column (Amersham Biosciences). Column-bound fusion proteins were digested overnight at 4 oC with 10 IU of Factor Xa (Amersham Biosciences), collected and stored at - 20 oC |
| Refolding Assay | Bioactivity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | |
| Purity | |
| Notes | |