Refolding Record:
| Protein | |
|---|---|
| Protein Name | Macrophage colony-stimulating factor |
| Abbreviated Name | MCSF |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P09603 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | n |
| Domain | aa.4-153 |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 154 |
| Molecular Weight | 17978.4 |
| Pi | 4.88326 |
| Molecular Weight | 17978.4 |
| Disulphides | 6 |
| Full Sequence |
EEVSEYCS HMIGSGHLQS LQRLIDSQME TSCQITFEFV DQEQLKDPVC YLKKAFLLVQ DIMEDTMRFR DNTPNAIAIV QLQELSLRLK SCFTKDYEEH DKACVRTFYE TPLQLLEKVK
NVFNETKNLL DKDWNIFSKN CNNSFAECSS QDVVTK
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Wilkins JA, Cone J, Randhawa ZI, Wood D, Warren MK, Witkowska HE (1993) Protein Science, 2, 244-254 |
| Project Aim | Folding |
| Fusion | None |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | SG21171 |
| Expression Temp | 42.0 |
| Expression Time | 3h |
| Expression Vector | not stated |
| Expression Protocol | Cells were fermented in a fed-batch mode. Protein production was induced by a change in temperature from 32 to 38-42degC. Induced cells were grown for 3h and then harvested either by centrifugation or tangential flow filtration. |
| Method of Induction | Temperature Shift |
| Cell Density at Induction | OD = |
| Cell Disruption Method | High pressure homogenization |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution |
| Wash Buffer | n/a |
| Solubilization Buffer | 5M urea, 20mM TrisCl, 50mM 2-mercaptoethanol pH 8.0 |
| Refolding Buffer | 50mM glycine pH 9.0 |
| Pre-Refolding Purification | None |
| Tag Cleaved | no tag |
| Refolding pH | 9.0 |
| Refolding Temperature | 25.0 |
| Protein Concentration | 200microg/ml |
| Refolding Time | 48h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | The cells were lysed in a homogenizer and centrifuged. The pelleted material was resuspended in solubilization buffer and then incubated for 2h at room temperature with mixing. Following this, a flocculating agent (BPA 1000) was added at 1000ppm to facilitate the removal of insoluble contaminants and nucleic acids. The suspension was centrifuged (5000g, 15min) and the supernatant was added directly into refolding buffer at a final monomeric protein concentration of 200microg/ml. Following a 48h incubation at room temperature, dimeric protein was purified. |
| Refolding Assay | HPLC |
| Refolding Chaperones | None |
| Refolding Additives | None,Glycine |
| Additives Concentration | 50mM |
| Refolding Yield | |
| Purity | |
| Notes | See also Randhawa et al., Biochemistry 1994, v33, 4352-4362 for protocol details |