Refolding Record:
| Protein | |
|---|---|
| Protein Name | Interleukin-2 |
| Abbreviated Name | IL-2 |
| SCOP Family | Short Chain Cytokines |
| Structure Notes | |
| Organism | Bovine |
| UniProt Accession | P05016 |
| 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 | 135 |
| Molecular Weight | 15510.0 |
| Pi | 5.74491 |
| Molecular Weight | 15510.0 |
| Disulphides | 1 |
| Full Sequence |
APTSSSTGNT MKEVKSLLLD LQLLLEKVKN PENLKLSRMH TFDFYVPKVN ATELKHLKCL LEELKLLEEV LNLAPSKNLN PREIKDSMDN IKRIVLELQG SETRFTCEYD DATVNAVEFL NKWITFCQSI YSTMT
|
| Notes | n/a |
| Expression | |
|---|---|
| Report | Kashima T, Morishita A, Iwata H, Maeda K, Inoue T (1999) J Vet Med B Infect Dis Vet Public Health, 61, 171-173 |
| Project Aim | Functional Studies |
| Fusion | N-terminal GST |
| Protein Expression and Production | Protein recombinantly expressed as and refolded from inclusion bodies. |
| Expression Host | Escherichia coli |
| Expression Strain | None |
| Expression Temp | 37.0 |
| Expression Time | not stated |
| Expression Vector | pGEX-5X-1 |
| Expression Protocol | 200ml of 2TY broth containing 20g/l glucose was inoculated with an overnight culture and grown at 37degC for 2h. Protein expression was induced wtih 0.1mM IPTG. Cells were harvested by centrifugation (7700g, 5min, 4degC), resuspended in ice cold PBS and sonicated. |
| Method of Induction | IPTG |
| Cell Density at Induction | OD = |
| Cell Disruption Method | Sonication |
| Lytic Agent | None |
| Pre-Refolding Purification | None |
| Solubility | insoluble |
| Refolding | |
|---|---|
| Refolding Method | Dialysis |
| Wash Buffer | n/a |
| Solubilization Buffer | 8M urea, 50mM TrisHCl pH 8.0, 1mM DTT, 1mM EDTA |
| Refolding Buffer | 50mM TrisHCl pH 8.0, 1mM DTT |
| Pre-Refolding Purification | None |
| Tag Cleaved | yes |
| Refolding pH | 8.0 |
| Refolding Temperature | 4.0 |
| Protein Concentration | |
| Refolding Time | overnight |
| Redox Agent | DTT |
| Redox Agent Concentration | 1mM |
| Refolding Protocol | The inclusion body pellet was resuspended in 10ml solubilization buffer and incubated for 1h at room temperature. The supernatant was dialyzed twice against 100ml of 4M urea, then 2M urea for 1hr each round at 4degC. The protein was then dialysed against 100ml refolding buffer wfor 1h at 4degC twice, then overnight against another 100ml of the same buffer. The protein was centrifuged and the supernatant retained. The protein was then purified using glutathione sepharose 4B. Cleavage of the GST tag was attempted by on-column cleavage using Factor Xa |
| Refolding Assay | SDS-PAGE |
| Refolding Chaperones | None |
| Refolding Additives | None |
| Additives Concentration | |
| Refolding Yield | |
| Purity | |
| Notes | |