Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-5 |
| Abbreviated Name | IL-5 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Human |
| UniProt Accession | P05113 |
| SCOP Unique ID | n/a |
| Structure Solved | |
| Class | Alpha |
| Molecularity | Dimer |
| Construct | |
|---|---|
| Full Length | y |
| Domain | n/a |
| Chimera | n/a |
| Variants | n/a |
| Chain Length | 136 |
| Molecular Weight | 15237.8 |
| Pi | 7.80915 |
| Molecular Weight | 15237.8 |
| Disulphides | 2 |
| Full Sequence |
MRMLLHLSLLALGAAYVYAIPTEIPTSALVKETLALLSTHRTLLIANETLRIPVPVHKNH
QLCTEEIFQGIGTLESQTVQGGTVERLFKNLSLIKKYIDGQKKKCGEERRRVNQFLDYLQ
EFLGVMNTEWIIES
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Mehta DV, DiGate RJ, Banville DL, Guiles RD. (1997) Protein Expression and Purification, 11, 86-94 |
| Project Aim | Undefined |
| Fusion | None |
| 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 | 4h |
| Expression Vector | pET11a |
| Expression Protocol | unknown |
| Method of Induction | Not Stated |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication/French Press |
| Lytic Agent | None |
| Pre-Refolding Purification | Ammonium Sulfate precipitation/gel filtration |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dilution/Dialysis combination |
| Wash Buffer | 2M urea, 0.1M Tris-HCl, 5mM benzamidine, 0.5mM PMSF, 1mM EDTA, 1mM DTT, pH 8.0 |
| Solubilization Buffer | 20mM Tris-HCl, 8M urea, 5mM EDTA, 1mM DTT, pH 8.5 |
| Refolding Buffer | 0.1 M Tris?HCl, 2M urea |
| Pre-Refolding Purification | Ammonium Sulfate precipitation/gel filtration |
| Tag Cleaved | no tag |
| Refolding pH | 8.5 |
| Refolding Temperature | 4.0 |
| Protein Concentration | 0.06 mg/ml |
| Refolding Time | 24h |
| Redox Agent | None |
| Redox Agent Concentration | n/a |
| Refolding Protocol | Solid ammonium sulfate was added to solubilized inclusion bodies to a level of 25% saturation and the precipitate was collected by centrifugation. The precipitate was dissolved in 10?5 ml of buffer containing 0.1 M Tris?HCl, pH 8.0, 6 M guanidinium chloride, 5 mM EDTA, 1 mM DTT and heated for 1 h at 60C to solubilize the inclusion bodies. After cooling to room temperature, the solution was applied to a Sephacryl S-200 column... |
| Refolding Assay | Enzyme activity |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | NULL |
| Refolding Yield | 10-12mg/L culture |
| Purity | 80% |
| Notes | |